P5CRs as modifiers of the p53 pathway and methods of use

ABSTRACT

Human P5CR genes are identified as modulators of the p53 pathway, and thus are therapeutic targets for disorders associated with defective p53 function. Methods for identifying modulators of p53, comprising screening for agents that modulate the activity of P5CR are provided.

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional patentapplications No. 60/296,080 filed Jun. 5, 2001, and No. 60/328,509,filed Oct. 10, 2001. The contents of the prior applications are herebyincorporated in their entirety.

BACKGROUND OF THE INVENTION

[0002] The p53 gene is mutated in over 50 different types of humancancers, including familial and spontaneous cancers, and is believed tobe the most commonly mutated gene in human cancer (Zambetti and Levine,FASEB (1993) 7:855-865; Hollstein, et al., Nucleic Acids Res. (1994)22:3551-3555). Greater than 90% of mutations in the p53 gene aremissense mutations that alter a single amino acid that inactivates p53function. Aberrant forms of human p53 are associated with poorprognosis, more aggressive tumors, metastasis, and short survival rates(Mitsudomi et al., Clin Cancer Res 2000 Oct; 6(10):4055-63; Koshland,Science (1993) 262:1953).

[0003] The human p53 protein normally functions as a central integratorof signals including DNA damage, hypoxia, nucleotide deprivation, andoncogene activation (Prives, Cell (1998) 95:5-8). In response to thesesignals, p53 protein levels are greatly increased with the result thatthe accumulated p53 activates cell cycle arrest or apoptosis dependingon the nature and strength of these signals. Indeed, multiple lines ofexperimental evidence have pointed to a key role for p53 as a tumorsuppressor (Levine, Cell (1997) 88:323-331). For example, homozygous p53“knockout” mice are developmentally normal but exhibit nearly 100%incidence of neoplasia in the first year of life (Donehower et al.,Nature (1992) 356:215-221).

[0004] The biochemical mechanisms and pathways through which p53functions in normal and cancerous cells are not fully understood, butone clearly important aspect of p53 function is its activity as agene-specific transcriptional activator. Among the genes with knownp53-response elements are several with well-characterized roles ineither regulation of the cell cycle or apoptosis, including GADD45,p21/Waf1/Cip1, cyclin G, Bax, IGF-BP3, and MDM2 (Levine, Cell (1997)88:323-331).

[0005] Pyrroline 5 carboxylate reductase (P5CR) catalyzes theNAD(P)H-dependent conversion of Pyrroline 5 carboxylate (P5C) toproline. Proline is itself oxidized to P5C by proline oxidase (PUT1) inmitochondria. P5C is further oxidized in mitochondria by P5Cdehydrogenase (PUT2) to glutamate.

[0006] Accumulation of P5C is implicated in p53-dependent initiation ofapoptosis. Proline oxidase is up-regulated in carcinoma cell lineschallenged with a p53-expressing adenovirus, leading to apoptosis of thecells. Moreover, cell-permeable conjugates of P5C alone induce apoptosis(Maxwell and Davis 2000 Proc Natl Acad Sci USA 97:13009-14). To date,however, a direct genetic relationship between p53 and P5CR has not beenestablished. P5CR is overexpressed in tumors of the colon, liver, andlung (Herzfeld A, et al., 1978 Cancer 42:1280-1283; Herzfeld A, et al.,1980 Cancer 45:2383-2388; Herzfeld A, and Greengard O. 1980 Cancer46:2047-2054; Notetrman D A et al. (2001) Cancer Res 61:3124-3130).

[0007] DNA and protein sequences for P5CR have been identified in a widevariety of organisms, including the yeast (DNA Genbank Identifier number(GI#) M57886; protein: GI# AAA34905), arabidopsis (DNA GI#977548;protein GI# CAC01879), Drosophila (DNA GI#5733737; protein GI#5733740),mouse (DNA GI#13879493; protein GI#13879494); and human (DNA GI#s189497and 10435995; Protein GI#s 189498 and 10435996), among others.

[0008] The ability to manipulate the genomes of model organisms such asDrosophila and provides a powerful means to analyze biochemicalprocesses that, due to significant evolutionary conservation, has directrelevance to more complex vertebrate organisms. Due to a high level ofgene and pathway conservation, the strong similarity of cellularprocesses, and the functional conservation of genes between these modelorganisms and mammals, identification of the involvement of novel genesin particular pathways and their functions in such model organisms candirectly contribute to the understanding of the correlative pathways andmethods of modulating them in mammals (see, for example, Mechler B M etal., 1985 EMBO J 4:1551-1557; Gateff E. 1982 Adv. Cancer Res. 37: 33-74;Watson K L., et al., 1994 J Cell Sci. 18: 19-33; Miklos G L, and Rubin GM. 1996 Cell 86:521-529; Wassarman D A, et al., 1995 Curr Opin Gen Dev5: 44-50; and Booth D R. 1999 Cancer Metastasis Rev. 18: 261-284). Forexample, a genetic screen can be carried out in an invertebrate modelorganism having underexpression (e.g. knockout) or overexpression of agene (referred to as a “genetic entry point”) that yields a visiblephenotype. Additional genes are mutated in a random or targeted manner.When a gene mutation changes the original phenotype caused by themutation in the genetic entry point, the gene is identified as a“modifier” involved in the same or overlapping pathway as the geneticentry point. When the genetic entry point is an ortholog of a human geneimplicated in a disease pathway, such as p53, modifier genes can beidentified that may be attractive candidate targets for noveltherapeutics.

[0009] All references cited herein, including sequence information inreferenced Genbank identifier numbers and website references, areincorporated herein in their entireties.

SUMMARY OF THE INVENTION

[0010] We have discovered genes that modify the p53 pathway inDrosophila, and identified their human orthologs, hereinafter referredto as P5CR. The invention provides methods for utilizing these p53modifier genes and polypeptides to identify candidate therapeutic agentsthat can be used in the treatment of disorders associated with defectivep53 function. Preferred P5CR-modulating agents specifically bind to P5CRpolypeptides and restore p53 function. Other preferred P5CR-modulatingagents are nucleic acid modulators such as antisense oligomers and RNAithat repress P5CR gene expression or product activity by, for example,binding to and inhibiting the respective nucleic acid (i.e. DNA ormRNA).

[0011] P5CR-specific modulating agents may be evaluated by anyconvenient in vitro or in vivo assay for molecular interaction with aP5CR polypeptide or nucleic acid. In one embodiment, candidate p53modulating agents are tested with an assay system comprising a P5CRpolypeptide or nucleic acid. Candidate agents that produce a change inthe activity of the assay system relative to controls are identified ascandidate p53 modulating agents. The assay system may be cell-based orcell-free. P5CR-modulating agents include P5CR related proteins (e.g.dominant negative mutants, and biotherapeutics); P5CR-specificantibodies; P5CR-specific antisense oligomers and other nucleic acidmodulators; and chemical agents that specifically bind P5CR or competewith P5CR binding target. In one specific embodiment, a small moleculemodulator is identified using an enzymatic assay. In specificembodiments, the screening assay system is selected from an apoptosisassay, a cell proliferation assay, an angiogenesis assay, and a hypoxicinduction assay.

[0012] In another embodiment, candidate p53 pathway modulating agentsare further tested using a second assay system that detects changes inthe p53 pathway, such as angiogenic, apoptotic, or cell proliferationchanges produced by the originally identified candidate agent or anagent derived from the original agent. The second assay system may usecultured cells or non-human animals. In specific embodiments, thesecondary assay system uses non-human animals, including animalspredetermined to have a disease or disorder implicating the p53 pathway,such as an angiogenic, apoptotic, or cell proliferation disorder (e.g.cancer).

[0013] The invention further provides methods for modulating the p53pathway in a mammalian cell by contacting the mammalian cell with anagent that specifically binds a P5CR polypeptide or nucleic acid. Theagent may be a small molecule modulator, a nucleic acid modulator, or anantibody and may be administered to a mammalian animal predetermined tohave a pathology associated the p53 pathway.

DETAILED DESCRIPTION OF THE INVENTION

[0014] Genetic modifier screens were designed to identify modifiers ofthe p53 pathway in Drosophila where p53 was overexpressed in the wing(Ollmann M, et al., Cell 2000 101: 91-101). The Drosophila P5CR gene wasidentified as a modifier of the p53 pathway. Accordingly, vertebrateorthologs of these modifiers, and preferably the human orthologs, P5CRgenes (i.e., nucleic acids and polypeptides) are attractive drug targetsfor the treatment of pathologies associated with a defective p53signaling pathway, such as cancer.

[0015] In vitro and in vivo methods of assessing P5CR function areprovided herein. Modulation of the P5CR or their respective bindingpartners is useful for understanding the association of the p53 pathwayand its members in normal and disease conditions and for developingdiagnostics and therapeutic modalities for p53 related pathologies.P5CR-modulating agents that act by inhibiting or enhancing P5CRexpression, directly or indirectly, for example, by affecting a P5CRfunction such as enzymatic (e.g., catalytic) or binding activity, can beidentified using methods provided herein. Inhibiting P5CR function mayinduce apoptosis or enhance activity of apoptosis-inducing agents. Thus,P5CR modulating agents are useful in diagnosis, therapy andpharmaceutical development.

[0016] Nucleic Acids and Polypeptides of the Invention

[0017] Sequences related to P5CR nucleic acids and polypeptides that canbe used in the invention are disclosed in Genbank (referenced by Genbankidentifier (GI) number), as GI#s 189497 (SEQ ID NO:1), 10435995 (SEQ IDNO:2), 16306657 (SEQ ID NO: 3), 18391546 (SEQ ID NO:4), 14124939 (SEQ IDNO:7), 18089015 (SEQ ID NO:8), and 4960117 (SEQ ID NO:9) for nucleicacid, and GI#s 189498 (SEQ ID NO:10), 10435996 (SEQ ID NO:11), 5902036(SEQ ID NO:12), 12751493 (SEQ ID NO:13), and 7019479 (SEQ ID NO:14) forpolypeptides.

[0018] P5CRs are reductase proteins with P5CR domains. The term “P5CRpolypeptide” refers to a full-length P5CR protein or a functionallyactive fragment or derivative thereof. A “functionally active” P5CRfragment or derivative exhibits one or more functional activitiesassociated with a full-length, wild-type P5CR protein, such as antigenicor immunogenic activity, enzymatic activity, ability to bind naturalcellular substrates, etc. The functional activity of P5CR proteins,derivatives and fragments can be assayed by various methods known to oneskilled in the art (Current Protocols in Protein Science (1998) Coliganet al., eds., John Wiley & Sons, Inc., Somerset, N.J.) and as furtherdiscussed below. For purposes herein, functionally active fragments alsoinclude those fragments that comprise one or more structural domains ofa P5CR, such as a reductase domain or a binding domain. Protein domainscan be identified using the PFAM program (Bateman A., et al., NucleicAcids Res, 1999, 27:260-2; http://pfam.wust1.edu). For example, P5CRdomain of SEQ ID NO:10 is located at amino acids 1-253 (PFAM 01089).Methods for obtaining P5CR polypeptides are also further describedbelow. In some embodiments, preferred fragments are functionally active,domain-containing fragments comprising at least 25 contiguous aminoacids, preferably at least 50, more preferably 75, and most preferablyat least 100 contiguous amino acids of any one of SEQ ID NOs:10, 11, 12,13, or 14 (a P5CR). In further preferred embodiments, the fragmentcomprises the entire reductase (functionally active) domain.

[0019] The term “P5CR nucleic acid” refers to a DNA or RNA molecule thatencodes a P5CR polypeptide. Preferably, the P5CR polypeptide or nucleicacid or fragment thereof is from a human, but can also be an ortholog,or derivative thereof with at least 70% sequence identity, preferably atleast 80%, more preferably 85%, still more preferably 90%, and mostpreferably at least 95% sequence identity with P5CR. As used herein,“percent (%) sequence identity” with respect to a subject sequence, or aspecified portion of a subject sequence, is defined as the percentage ofnucleotides or amino acids in the candidate derivative sequenceidentical with the nucleotides or amino acids in the subject sequence(or specified portion thereof), after aligning the sequences andintroducing gaps, if necessary to achieve the maximum percent sequenceidentity, as generated by the program WU-BLAST-2.0a19 (Altschul et al.,J. Mol. Biol. (1997) 215:403-410;http://blast.wust1.edu/blast/README.html) with all the search parametersset to default values. The HSP S and HSP S2 parameters are dynamicvalues and are established by the program itself depending upon thecomposition of the particular sequence and composition of the particulardatabase against which the sequence of interest is being searched. A %identity value is determined by the number of matching identicalnucleotides or amino acids divided by the sequence length for which thepercent identity is being reported. “Percent (%) amino acid sequencesimilarity” is determined by doing the same calculation as fordetermining % amino acid sequence identity, but including conservativeamino acid substitutions in addition to identical amino acids in thecomputation.

[0020] A conservative amino acid substitution is one in which an aminoacid is substituted for another amino acid having similar propertiessuch that the folding or activity of the protein is not significantlyaffected. Aromatic amino acids that can be substituted for each otherare phenylalanine, tryptophan, and tyrosine; interchangeable hydrophobicamino acids are leucine, isoleucine, methionine, and valine;interchangeable polar amino acids are glutamine and asparagine;interchangeable basic amino acids are arginine, lysine and histidine;interchangeable acidic amino acids are aspartic acid and glutamic acid;and interchangeable small amino acids are alanine, serine, threonine,cysteine and glycine.

[0021] Alternatively, an alignment for nucleic acid sequences isprovided by the local homology algorithm of Smith and Waterman (Smithand Waterman, 1981, Advances in Applied Mathematics 2:482-489; database:European Bioinformatics Institute http://www.ebi.ac.uk/MPsrch/; Smithand Waterman, 1981, J. of Molec. Biol., 147:195-197; Nicholas et al.,1998, “A Tutorial on Searching Sequence Databases and Sequence ScoringMethods” (www.psc.edu) and references cited therein.; W. R. Pearson,1991, Genomics 11:635-650). This algorithm can be applied to amino acidsequences by using the scoring matrix developed by Dayhoff (Dayhoff:Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl.3:353-358, National Biomedical Research Foundation, Washington, D.C.,USA), and normalized by Gribskov (Gribskov 1986 Nucl. Acids Res.14(6):6745-6763). The Smith-Waterman algorithm may be employed wheredefault parameters are used for scoring (for example, gap open penaltyof 12, gap extension penalty of two). From the data generated, the“Match” value reflects “sequence identity.”

[0022] Derivative nucleic acid molecules of the subject nucleic acidmolecules include sequences that hybridize to the nucleic acid sequenceof SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, or 9. The stringency ofhybridization can be controlled by temperature, ionic strength, pH, andthe presence of denaturing agents such as formamide during hybridizationand washing. Conditions routinely used are set out in readily availableprocedure texts (e.g., Current Protocol in Molecular Biology, Vol. 1,Chap. 2.10, John Wiley & Sons, Publishers (1994); Sambrook et al.,Molecular Cloning, Cold Spring Harbor (1989)). In some embodiments, anucleic acid molecule of the invention is capable of hybridizing to anucleic acid molecule containing the nucleotide sequence of any one ofSEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, or 9 under stringent hybridizationconditions that comprise: prehybridization of filters containing nucleicacid for 8 hours to overnight at 65° C. in a solution comprising 6×single strength citrate (SSC) (1× SSC is 0.15 M NaCl, 0.015 M Nacitrate; pH 7.0), 5× Denhardt's solution, 0.05% sodium pyrophosphate and100 μg/ml herring sperm DNA; hybridization for 18-20 hours at 65° C. ina solution containing 6× SSC, 1× Denhardt's solution, 100 μg/ml yeasttRNA and 0.05% sodium pyrophosphate; and washing of filters at 65° C.for 1 h in a solution containing 0.2× SSC and 0.1% SDS (sodium dodecylsulfate).

[0023] In other embodiments, moderately stringent hybridizationconditions are used that comprise: pretreatment of filters containingnucleic acid for 6 h at 40° C. in a solution containing 35% formamide,5× SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1%BSA, and 500 μg/ml denatured salmon sperm DNA; hybridization for 18-20 hat 40° C. in a solution containing 35% formamide, 5× SSC, 50 mM Tris-HCl(pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmonsperm DNA, and 10% (wt/vol) dextran sulfate; followed by washing twicefor 1 hour at 55° C. in a solution containing 2× SSC and 0.1% SDS.

[0024] Alternatively, low stringency conditions can be used thatcomprise: incubation for 8 hours to overnight at 37° C. in a solutioncomprising 20% formamide, 5× SSC, 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured shearedsalmon sperm DNA; hybridization in the same buffer for 18 to 20 hours;and washing of filters in 1× SSC at about 37° C. for 1 hour.

[0025] Isolation, Production, Expression, and Mis-Expression of P5CRNucleic Acids and Polypeptides

[0026] P5CR nucleic acids and polypeptides, useful for identifying andtesting agents that modulate P5CR function and for other applicationsrelated to the involvement of P5CR in the p53 pathway. P5CR nucleicacids and derivatives and orthologs thereof may be obtained using anyavailable method. For instance, techniques for isolating cDNA or genomicDNA sequences of interest by screening DNA libraries or by usingpolymerase chain reaction (PCR) are well known in the art. In general,the particular use for the protein will dictate the particulars ofexpression, production, and purification methods. For instance,production of proteins for use in screening for modulating agents mayrequire methods that preserve specific biological activities of theseproteins, whereas production of proteins for antibody generation mayrequire structural integrity of particular epitopes. Expression ofproteins to be purified for screening or antibody production may requirethe addition of specific tags (e.g., generation of fusion proteins).Overexpression of a P5CR protein for assays used to assess P5CRfunction, such as involvement in cell cycle regulation or hypoxicresponse, may require expression in eukaryotic cell lines capable ofthese cellular activities. Techniques for the expression, production,and purification of proteins are well known in the art; any suitablemeans therefore may be used (e.g., Higgins S J and Hames B D (eds.)Protein Expression: A Practical Approach, Oxford University Press Inc.,New York 1999; Stanbury P F et al., Principles of FermentationTechnology, 2^(nd) edition, Elsevier Science, New York, 1995; Doonan S(ed.) Protein Purification Protocols, Humana Press, New Jersey, 1996;Coligan J E et al, Current Protocols in Protein Science (eds.), 1999,John Wiley & Sons, New York). In particular embodiments, recombinantP5CR is expressed in a cell line known to have defective p53 function(e.g. SAOS-2 osteoblasts, H1299 lung cancer cells, C33A and HT3 cervicalcancer cells, HT-29 and DLD-1 colon cancer cells, among others,available from American Type Culture Collection (ATCC), Manassas, Va.).The recombinant cells are used in cell-based screening assay systems ofthe invention, as described further below.

[0027] The nucleotide sequence encoding a P5CR polypeptide can beinserted into any appropriate expression vector. The necessarytranscriptional and translational signals, including promoter/enhancerelement, can derive from the native P5CR gene and/or its flankingregions or can be heterologous. A variety of host-vector expressionsystems may be utilized, such as mammalian cell systems infected withvirus (e.g. vaccinia virus, adenovirus, etc.); insect cell systemsinfected with virus (e.g. baculovirus); microorganisms such as yeastcontaining yeast vectors, or bacteria transformed with bacteriophage,plasmid, or cosmid DNA. A host cell strain that modulates the expressionof, modifies, and/or specifically processes the gene product may beused.

[0028] To detect expression of the P5CR gene product, the expressionvector can comprise a promoter operably linked to a P5CR gene nucleicacid, one or more origins of replication, and, one or more selectablemarkers (e.g. thymidine kinase activity, resistance to antibiotics,etc.). Alternatively, recombinant expression vectors can be identifiedby assaying for the expression of the P5CR gene product based on thephysical or functional properties of the P5CR protein in in vitro assaysystems (e.g. immunoassays).

[0029] The P5CR protein, fragment, or derivative may be optionallyexpressed as a fusion, or chimeric protein product (i.e. it is joinedvia a peptide bond to a heterologous protein sequence of a differentprotein), for example to facilitate purification or detection. Achimeric product can be made by ligating the appropriate nucleic acidsequences encoding the desired amino acid sequences to each other usingstandard methods and expressing the chimeric product. A chimeric productmay also be made by protein synthetic techniques, e.g. by use of apeptide synthesizer (Hunkapiller et al., Nature (1984) 310:105-111).

[0030] Once a recombinant cell that expresses the P5CR gene sequence isidentified, the gene product can be isolated and purified using standardmethods (e.g. ion exchange, affinity, and gel exclusion chromatography;centrifugation; differential solubility; electrophoresis, citepurification reference). Alternatively, native P5CR proteins can bepurified from natural sources, by standard methods (e.g. immunoaffinitypurification). Once a protein is obtained, it may be quantified and itsactivity measured by appropriate methods, such as immunoassay, bioassay,or other measurements of physical properties, such as crystallography.

[0031] The methods of this invention may also use cells that have beenengineered for altered expression (mis-expression) of P5CR or othergenes associated with the p53 pathway. As used herein, mis-expressionencompasses ectopic expression, over-expression, under-expression, andnon-expression (e.g. by gene knock-out or blocking expression that wouldotherwise normally occur).

[0032] Genetically Modified Animals

[0033] Animal models that have been genetically modified to alter P5CRexpression may be used in in vivo assays to test for activity of acandidate p53 modulating agent, or to further assess the role of P5CR ina p53 pathway process such as apoptosis or cell proliferation.Preferably, the altered P5CR expression results in a detectablephenotype, such as decreased or increased levels of cell proliferation,angiogenesis, or apoptosis compared to control animals having normalP5CR expression. The genetically modified animal may additionally havealtered p53 expression (e.g. p53 knockout). Preferred geneticallymodified animals are mammals such as primates, rodents (preferablymice), cows, horses, goats, sheep, pigs, dogs and cats. Preferrednon-mammalian species include zebrafish, C. elegans, and Drosophila.Preferred genetically modified animals are transgenic animals having aheterologous nucleic acid sequence present as an extrachromosomalelement in a portion of its cells, i.e. mosaic animals (see, forexample, techniques described by Jakobovits, 1994, Curr. Biol.4:761-763.) or stably integrated into its germ line DNA (i.e., in thegenomic sequence of most or all of its cells). Heterologous nucleic acidis introduced into the germ line of such transgenic animals by geneticmanipulation of, for example, embryos or embryonic stem cells of thehost animal.

[0034] Methods of making transgenic animals are well-known in the art(for transgenic mice see Brinster et al., Proc. Nat. Acad. Sci. USA 82:4438-4442 (1985), U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Lederet al., U.S. Pat. No. 4,873,191 by Wagner et al., and Hogan, B.,Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986); for particle bombardment see U.S. Pat. No.,4,945,050, by Sandford et al.; for transgenic Drosophila see Rubin andSpradling, Science (1982) 218:348-53 and U.S. Pat. No. 4,670,388; fortransgenic insects see Berghammer A. J. et al., A Universal Marker forTransgenic Insects (1999) Nature 402:370-371; for transgenic Zebrafishsee Lin S., Transgenic Zebrafish, Methods Mol Biol.(2000);136:375-3830); for microinjection procedures for fish, amphibianeggs and birds see Houdebine and Chourrout, Experientia (1991)47:897-905; for transgenic rats see Hammer et al., Cell (1990)63:1099-1112; and for culturing of embryonic stem (ES) cells and thesubsequent production of transgenic animals by the introduction of DNAinto ES cells using methods such as electroporation, calciumphosphate/DNA precipitation and direct injection see, e.g.,Teratocarcinomas and Embryonic Stem Cells, A Practical Approach, E. J.Robertson, ed., IRL Press (1987)). Clones of the nonhuman transgenicanimals can be produced according to available methods (see Wilmut, I.et al. (1997) Nature 385:810-813; and PCT International Publication Nos.WO 97/07668 and WO 97/07669).

[0035] In one embodiment, the transgenic animal is a “knock-out” animalhaving a heterozygous or homozygous alteration in the sequence of anendogenous P5CR gene that results in a decrease of P5CR function,preferably such that P5CR expression is undetectable or insignificant.Knock-out animals are typically generated by homologous recombinationwith a vector comprising a transgene having at least a portion of thegene to be knocked out. Typically a deletion, addition or substitutionhas been introduced into the transgene to functionally disrupt it. Thetransgene can be a human gene (e.g., from a human genomic clone) butmore preferably is an ortholog of the human gene derived from thetransgenic host species. For example, a mouse P5CR gene is used toconstruct a homologous recombination vector suitable for altering anendogenous P5CR gene in the mouse genome. Detailed methodologies forhomologous recombination in mice are available (see Capecchi, Science(1989) 244:1288-1292; Joyner et al., Nature (1989) 338:153-156).Procedures for the production of non-rodent transgenic mammals and otheranimals are also available (Houdebine and Chourrout, supra; Pursel etal., Science (1989) 244:1281-1288; Simms et al., Bio/Technology (1988)6:179-183). In a preferred embodiment, knock-out animals, such as miceharboring a knockout of a specific gene, may be used to produceantibodies against the human counterpart of the gene that has beenknocked out (Claesson M H et al., (1994) Scan J Immunol 40:257-264;Declerck P J et al., (1995) J Biol Chem. 270:8397-400).

[0036] In another embodiment, the transgenic animal is a “knock-in”animal having an alteration in its genome that results in alteredexpression (e.g., increased (including ectopic) or decreased expression)of the P5CR gene, e.g., by introduction of additional copies of P5CR, orby operatively inserting a regulatory sequence that provides for alteredexpression of an endogenous copy of the P5CR gene. Such regulatorysequences include inducible, tissue-specific, and constitutive promotersand enhancer elements. The knock-in can be homozygous or heterozygous.

[0037] Transgenic nonhuman animals can also be produced that containselected systems allowing for regulated expression of the transgene. Oneexample of such a system that may be produced is the cre/loxPrecombinase system of bacteriophage P1 (Lakso et al., PNAS (1992)89:6232-6236; U.S. Pat. No. 4,959,317). If a cre/loxP recombinase systemis used to regulate expression of the transgene, animals containingtransgenes encoding both the Cre recombinase and a selected protein arerequired. Such animals can be provided through the construction of“double” transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase. Another example of arecombinase system is the FLP recombinase system of Saccharomycescerevisiae (O'Gorman et al. (1991) Science 251:1351-1355; U.S. Pat. No.5,654,182). In a preferred embodiment, both Cre-LoxP and Flp-Frt areused in the same system to regulate expression of the transgene, and forsequential deletion of vector sequences in the same cell (Sun X et al(2000) Nat Genet 25:83-6).

[0038] The genetically modified animals can be used in genetic studiesto further elucidate the p53 pathway, as animal models of disease anddisorders implicating defective p53 function, and for in vivo testing ofcandidate therapeutic agents, such as those identified in screensdescribed below. The candidate therapeutic agents are administered to agenetically modified animal having altered P5CR function and phenotypicchanges are compared with appropriate control animals such asgenetically modified animals that receive placebo treatment, and/oranimals with unaltered P5CR expression that receive candidatetherapeutic agent.

[0039] In addition to the above-described genetically modified animalshaving altered P5CR function, animal models having defective p53function (and otherwise normal P5CR function), can be used in themethods of the present invention. For example, a p53 knockout mouse canbe used to assess, in vivo, the activity of a candidate p53 modulatingagent identified in one of the in vitro assays described below. p53knockout mice are described in the literature (Jacks et al., Nature2001;410:1111-1116, 1043-1044; Donehower et al., supra). Preferably, thecandidate p53 modulating agent when administered to a model system withcells defective in p53 function, produces a detectable phenotypic changein the model system indicating that the p53 function is restored, i.e.,the cells exhibit normal cell cycle progression.

[0040] Modulating Agents

[0041] The invention provides methods to identify agents that interactwith and/or modulate the function of P5CR and/or the p53 pathway. Suchagents are useful in a variety of diagnostic and therapeuticapplications associated with the p53 pathway, as well as in furtheranalysis of the P5CR protein and its contribution to the p53 pathway.Accordingly, the invention also provides methods for modulating the p53pathway comprising the step of specifically modulating P5CR activity byadministering a P5CR-interacting or -modulating agent.

[0042] In a preferred embodiment, P5CR-modulating agents inhibit orenhance P5CR activity or otherwise affect normal P5CR function,including transcription, protein expression, protein localization, andcellular or extra-cellular activity. In a further preferred embodiment,the candidate p53 pathway-modulating agent specifically modulates thefunction of the P5CR. The phrases “specific modulating agent”,“specifically modulates”, etc., are used herein to refer to modulatingagents that directly bind to the P5CR polypeptide or nucleic acid, andpreferably inhibit, enhance, or otherwise alter, the function of theP5CR. The term also encompasses modulating agents that alter theinteraction of the P5CR with a binding partner or substrate (e.g. bybinding to a binding partner of a P5CR, or to a protein/binding partnercomplex, and inhibiting function).

[0043] Preferred P5CR-modulating agents include small moleculecompounds; P5CR-interacting proteins; and nucleic acid modulators suchas antisense and RNA inhibitors. The modulating agents may be formulatedin pharmaceutical compositions, for example, as compositions that maycomprise other active ingredients, as in combination therapy, and/orsuitable carriers or excipients. Techniques for formulation andadministration of the compounds may be found in “Remington'sPharmaceutical Sciences” Mack Publishing Co., Easton, Pa., 19^(th)edition.

[0044] Small Molecule Modulators

[0045] Small molecules, are often preferred to modulate function ofproteins with enzymatic function, and/or containing protein interactiondomains. Chemical agents, referred to in the art as “small molecule”compounds are typically organic, non-peptide molecules, having amolecular weight less than 10,000, preferably less than 5,000, morepreferably less than 1,000, and most preferably less than 500. Thisclass of modulators includes chemically synthesized molecules, forinstance, compounds from combinatorial chemical libraries. Syntheticcompounds may be rationally designed or identified based on known orinferred properties of the P5CR protein or may be identified byscreening compound libraries. Alternative appropriate modulators of thisclass are natural products, particularly secondary metabolites fromorganisms such as plants or fungi, which can also be identified byscreening compound libraries for P5CR-modulating activity. Methods forgenerating and obtaining compounds are well known in the art (SchreiberS L, Science (2000) 151: 1964-1969; Radmann J and Gunther J, Science(2000) 151:1947-1948).

[0046] Small molecule modulators identified from screening assays, asdescribed below, can be used as lead compounds from which candidateclinical compounds may be designed, optimized, and synthesized. Suchclinical compounds may have utility in treating pathologies associatedwith the p53 pathway. The activity of candidate small moleculemodulating agents may be improved several-fold through iterativesecondary functional validation, as further described below, structuredetermination, and candidate modulator modification and testing.Additionally, candidate clinical compounds are generated with specificregard to clinical and pharmacological properties. For example, thereagents may be derivatized and re-screened using in vitro and in vivoassays to optimize activity and minimize toxicity for pharmaceuticaldevelopment.

[0047] Protein Modulators

[0048] Specific P5CR-interacting proteins are useful in a variety ofdiagnostic and therapeutic applications related to the p53 pathway andrelated disorders, as well as in validation assays for otherP5CR-modulating agents. In a preferred embodiment, P5CR-interactingproteins affect normal P5CR function, including transcription, proteinexpression, protein localization, and cellular or extra-cellularactivity. In another embodiment, P5CR-interacting proteins are useful indetecting and providing information about the function of P5CR proteins,as is relevant to p53 related disorders, such as cancer (e.g., fordiagnostic means).

[0049] A P5CR-interacting protein may be endogenous, i.e. one thatnaturally interacts genetically or biochemically with a P5CR, such as amember of the P5CR pathway that modulates P5CR expression, localization,and/or activity. P5CR-modulators include dominant negative forms ofP5CR-interacting proteins and of P5CR proteins themselves. Yeasttwo-hybrid and variant screens offer preferred methods for identifyingendogenous P5CR-interacting proteins (Finley, R. L. et al. (1996) in DNACloning-Expression Systems: A Practical Approach, eds. Glover D. & HamesB. D (Oxford University Press, Oxford, England), pp. 169-203; Fashema SF et al., Gene (2000) 250:1-14; Drees B L Curr Opin Chem Biol (1999)3:64-70; Vidal M and Legrain P Nucleic Acids Res (1999) 27:919-29; andU.S. Pat. No. 5,928,868). Mass spectrometry is an alternative preferredmethod for the elucidation of protein complexes (reviewed in, e.g.,Pandley A and Mann M, Nature (2000) 405:837-846; Yates J R 3^(rd),Trends Genet (2000) 16:5-8).

[0050] A P5CR-interacting protein may be an exogenous protein, such as aP5CR-specific antibody or a T-cell antigen receptor (see, e.g., Harlowand Lane (1988) Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory; Harlow and Lane (1999) Using antibodies: a laboratorymanual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press).P5CR antibodies are further discussed below.

[0051] In preferred embodiments, a P5CR-interacting protein specificallybinds a P5CR protein. In alternative preferred embodiments, aP5CR-modulating agent binds a P5CR substrate, binding partner, orcofactor.

[0052] Antibodies

[0053] In another embodiment, the protein modulator is a P5CR specificantibody agonist or antagonist. The antibodies have therapeutic anddiagnostic utilities, and can be used in screening assays to identifyP5CR modulators. The antibodies can also be used in dissecting theportions of the P5CR pathway responsible for various cellular responsesand in the general processing and maturation of the P5CR.

[0054] Antibodies that specifically bind P5CR polypeptides can begenerated using known methods. Preferably the antibody is specific to amammalian ortholog of P5CR polypeptide, and more preferably, to humanP5CR. Antibodies may be polyclonal, monoclonal (mAbs), humanized orchimeric antibodies, single chain antibodies, Fab fragments,F(ab′).sub.2 fragments, fragments produced by a FAb expression library,anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments ofany of the above. Epitopes of P5CR which are particularly antigenic canbe selected, for example, by routine screening of P5CR polypeptides forantigenicity or by applying a theoretical method for selecting antigenicregions of a protein (Hopp and Wood (1981), Proc. Natl. Acad. Sci.U.S.A. 78:3824-28; Hopp and Wood, (1983) Mol. Immunol. 20:483-89;Sutcliffe et al., (1983) Science 219:660-66) to the amino acid sequenceshown in SEQ ID NOs: 10, 11, 12, 13, or 14. Monoclonal antibodies withaffinities of 10⁸ M⁻¹ preferably 10⁹ M⁻¹ to 10¹⁰ M⁻¹, or stronger can bemade by standard procedures as described (Harlow and Lane, supra; Goding(1986) Monoclonal Antibodies: Principles and Practice (2d ed) AcademicPress, New York; and U.S. Pat. Nos. 4,381,292; 4,451,570; and4,618,577). Antibodies may be generated against crude cell extracts ofP5CR or substantially purified fragments thereof. If P5CR fragments areused, they preferably comprise at least 10, and more preferably, atleast 20 contiguous amino acids of a P5CR protein. In a particularembodiment, P5CR-specific antigens and/or immunogens are coupled tocarrier proteins that stimulate the immune response. For example, thesubject polypeptides are covalently coupled to the keyhole limpethemocyanin (KLH) carrier, and the conjugate is emulsified in Freund'scomplete adjuvant, which enhances the immune response. An appropriateimmune system such as a laboratory rabbit or mouse is immunizedaccording to conventional protocols.

[0055] The presence of P5CR-specific antibodies is assayed by anappropriate assay such as a solid phase enzyme-linked immunosorbantassay (ELISA) using immobilized corresponding P5CR polypeptides. Otherassays, such as radioimmunoassays or fluorescent assays might also beused.

[0056] Chimeric antibodies specific to P5CR polypeptides can be madethat contain different portions from different animal species. Forinstance, a human immunoglobulin constant region may be linked to avariable region of a murine mAb, such that the antibody derives itsbiological activity from the human antibody, and its binding specificityfrom the murine fragment. Chimeric antibodies are produced by splicingtogether genes that encode the appropriate regions from each species(Morrison et al., Proc. Natl. Acad. Sci. (1984) 81:6851-6855; Neubergeret al., Nature (1984) 312:604-608; Takeda et al., Nature (1985)31:452-454). Humanized antibodies, which are a form of chimericantibodies, can be generated by grafting complementary-determiningregions (CDRs) (Carlos, T. M., J. M. Harlan. 1994. Blood 84:2068-2101)of mouse antibodies into a background of human framework regions andconstant regions by recombinant DNA technology (Riechmann L M, et al.,1988 Nature 323: 323-327). Humanized antibodies contain ˜10% murinesequences and ˜90% human sequences, and thus further reduce or eliminateimmunogenicity, while retaining the antibody specificities (Co MS, andQueen C. 1991 Nature 351: 501-501; Morrison S L. 1992 Ann. Rev. Immun.10:239-265). Humanized antibodies and methods of their production arewell-known in the art (U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,762,and 6,180,370).

[0057] P5CR-specific single chain antibodies which are recombinant,single chain polypeptides formed by linking the heavy and light chainfragments of the Fv regions via an amino acid bridge, can be produced bymethods known in the art (U.S. Pat. No. 4,946,778; Bird, Science (1988)242:423-426; Huston et al., Proc. Natl. Acad. Sci. USA (1988)85:5879-5883; and Ward et al., Nature (1989) 334:544-546).

[0058] Other suitable techniques for antibody production involve invitro exposure of lymphocytes to the antigenic polypeptides oralternatively to selection of libraries of antibodies in phage orsimilar vectors (Huse et al., Science (1989) 246:1275-1281). As usedherein, T-cell antigen receptors are included within the scope ofantibody modulators (Harlow and Lane, 1988, supra).

[0059] The polypeptides and antibodies of the present invention may beused with or without modification. Frequently, antibodies will belabeled by joining, either covalently or non-covalently, a substancethat provides for a detectable signal, or that is toxic to cells thatexpress the targeted protein (Menard S, et al., Int J. Biol Markers(1989) 4:131-134). A wide variety of labels and conjugation techniquesare known and are reported extensively in both the scientific and patentliterature. Suitable labels include radionuclides, enzymes, substrates,cofactors, inhibitors, fluorescent moieties, fluorescent emittinglanthanide metals, chemiluminescent moieties, bioluminescent moieties,magnetic particles, and the like (U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241). Also,recombinant immunoglobulins may be produced (U.S. Pat. No. 4,816,567).Antibodies to cytoplasmic polypeptides may be delivered and reach theirtargets by conjugation with membrane-penetrating toxin proteins (U.S.Pat. No. 6,086,900).

[0060] When used therapeutically in a patient, the antibodies of thesubject invention are typically administered parenterally, when possibleat the target site, or intravenously. The therapeutically effective doseand dosage regimen is determined by clinical studies. Typically, theamount of antibody administered is in the range of about 0.1 mg/kg- toabout 10 mg/kg of patient weight. For parenteral administration, theantibodies are formulated in a unit dosage injectable form (e.g.,solution, suspension, emulsion) in association with a pharmaceuticallyacceptable vehicle. Such vehicles are inherently nontoxic andnon-therapeutic. Examples are water, saline, Ringer's solution, dextrosesolution, and 5% human serum albumin. Nonaqueous vehicles such as fixedoils, ethyl oleate, or liposome carriers may also be used. The vehiclemay contain minor amounts of additives, such as buffers andpreservatives, which enhance isotonicity and chemical stability orotherwise enhance therapeutic potential. The antibodies' concentrationsin such vehicles are typically in the range of about 1 mg/ml to about 10mg/ml. Immunotherapeutic methods are further described in the literature(U.S. Pat. No. 5,859,206; WO0073469).

[0061] Nucleic Acid Modulators

[0062] Other preferred P5CR-modulating agents comprise nucleic acidmolecules, such as antisense oligomers or double stranded RNA (dsRNA),which generally inhibit P5CR activity. Preferred nucleic acid modulatorsinterfere with the function of the P5CR nucleic acid such as DNAreplication, transcription, translocation of the P5CR RNA to the site ofprotein translation, translation of protein from the P5CR RNA, splicingof the P5CR RNA to yield one or more mRNA species, or catalytic activitywhich may be engaged in or facilitated by the P5CR RNA.

[0063] In one embodiment, the antisense oligomer is an oligonucleotidethat is sufficiently complementary to a P5CR mRNA to bind to and preventtranslation, preferably by binding to the 5′ untranslated region.P5CR-specific antisense oligonucleotides, preferably range from at least6 to about 200 nucleotides. In some embodiments the oligonucleotide ispreferably at least 10, 15, or 20 nucleotides in length. In otherembodiments, the oligonucleotide is preferably less than 50, 40, or 30nucleotides in length. The oligonucleotide can be DNA or RNA or achimeric mixture or derivatives or modified versions thereof,single-stranded or double-stranded. The oligonucleotide can be modifiedat the base moiety, sugar moiety, or phosphate backbone. Theoligonucleotide may include other appending groups such as peptides,agents that facilitate transport across the cell membrane,hybridization-triggered cleavage agents, and intercalating agents.

[0064] In another embodiment, the antisense oligomer is a phosphothioatemorpholino oligomer (PMO). PMOs are assembled from four differentmorpholino subunits, each of which contain one of four genetic bases (A,C, G, or T) linked to a six-membered morpholine ring. Polymers of thesesubunits are joined by non-ionic phosphodiamidate intersubunit linkages.Details of how to make and use PMOs and other antisense oligomers arewell known in the art (e.g. see WO99/18193; Probst J C, AntisenseOligodeoxynucleotide and Ribozyme Design, Methods. (2000) 22(3):271-281;Summerton J, and Weller D. 1997 Antisense Nucleic Acid Drug Dev.:7:187-95; U.S. Pat. No. 5,235,033; and U.S. Pat No. 5,378,841).

[0065] Alternative preferred P5CR nucleic acid modulators aredouble-stranded RNA species mediating RNA interference (RNAi). RNAi isthe process of sequence-specific, post-transcriptional gene silencing inanimals and plants, initiated by double-stranded RNA (dsRNA) that ishomologous in sequence to the silenced gene. Methods relating to the useof RNAi to silence genes in C. elegans, Drosophila, plants, and humansare known in the art (Fire A, et al., 1998 Nature 391:806-811; Fire, A.Trends Genet. 15, 358-363 (1999); Sharp, P. A. RNA interference 2001.Genes Dev. 15, 485-490 (2001); Hammond, S. M., et al., Nature Rev.Genet. 2, 110-1119 (2001); Tuschl, T. Chem. Biochem. 2, 239-245 (2001);Hamilton, A. et al., Science 286, 950-952 (1999); Hammond, S. M., etal., Nature 404, 293-296 (2000); Zamore, P. D., et al., Cell 101, 25-33(2000); Bernstein, E., et al., Nature 409, 363-366 (2001); Elbashir, S.M., et al., Genes Dev. 15, 188-200 (2001); WO0129058; WO9932619;Elbashir S M, et al., 2001 Nature 411:494-498). Example VII details theuse of RNAi to knock down P5CR expression in cell lines.

[0066] Nucleic acid modulators are commonly used as research reagents,diagnostics, and therapeutics. For example, antisense oligonucleotides,which are able to inhibit gene expression with exquisite specificity,are often used to elucidate the function of particular genes (see, forexample, U.S. Pat. No. 6,165,790). Nucleic acid modulators are alsoused, for example, to distinguish between functions of various membersof a biological pathway. For example, antisense oligomers have beenemployed as therapeutic moieties in the treatment of disease states inanimals and man and have been demonstrated in numerous clinical trialsto be safe and effective (Milligan J F, et al, Current Concepts inAntisense Drug Design, J Med Chem. (1993) 36:1923-1937; Tonkinson J L etal., Antisense Oligodeoxynucleotides as Clinical Therapeutic Agents,Cancer Invest. (1996) 14:54-65). Accordingly, in one aspect of theinvention, a P5CR-specific nucleic acid modulator is used in an assay tofurther elucidate the role of the P5CR in the p53 pathway, and/or itsrelationship to other members of the pathway. In another aspect of theinvention, a P5CR-specific antisense oligomer is used as a therapeuticagent for treatment of p53-related disease states.

[0067] Assay Systems

[0068] The invention provides assay systems and screening methods foridentifying specific modulators of P5CR activity. As used herein, an“assay system” encompasses all the components required for performingand analyzing results of an assay that detects and/or measures aparticular event. In general, primary assays are used to identify orconfirm a modulator's specific biochemical or molecular effect withrespect to the P5CR nucleic acid or protein. In general, secondaryassays further assess the activity of a P5CR modulating agent identifiedby a primary assay and may confirm that the modulating agent affectsP5CR in a manner relevant to the p53 pathway. In some cases, P5CRmodulators will be directly tested in a secondary assay.

[0069] In a preferred embodiment, the screening method comprisescontacting a suitable assay system comprising a P5CR polypeptide with acandidate agent under conditions whereby, but for the presence of theagent, the system provides a reference activity (e.g. enzymaticactivity), which is based on the particular molecular event thescreening method detects. A statistically significant difference betweenthe agent-biased activity and the reference activity indicates that thecandidate agent modulates P5CR activity, and hence the p53 pathway.

[0070] Primary Assays

[0071] The type of modulator tested generally determines the type ofprimary assay.

[0072] Primary Assays for Small Molecule Modulators

[0073] For small molecule modulators, screening assays are used toidentify candidate modulators. Screening assays may be cell-based or mayuse a cell-free system that recreates or retains the relevantbiochemical reaction of the target protein (reviewed in Sittampalam G Set al., Curr Opin Chem Biol (1997) 1:384-91 and accompanyingreferences). As used herein the term “cell-based” refers to assays usinglive cells, dead cells, or a particular cellular fraction, such as amembrane, endoplasmic reticulum, or mitochondrial fraction. The term“cell free” encompasses assays using substantially purified protein(either endogenous or recombinantly produced), partially purified orcrude cellular extracts. Screening assays may detect a variety ofmolecular events, including protein-DNA interactions, protein-proteininteractions (e.g., receptor-ligand binding), transcriptional activity(e.g., using a reporter gene), enzymatic activity (e.g., via a propertyof the substrate), activity of second messengers, immunogenicty andchanges in cellular morphology or other cellular characteristics.Appropriate screening assays may use a wide range of detection methodsincluding fluorescent, radioactive, calorimetric, spectrophotometric,and amperometric methods, to provide a read-out for the particularmolecular event detected.

[0074] Cell-based screening assays usually require systems forrecombinant expression of P5CR and any auxiliary proteins demanded bythe particular assay. Appropriate methods for generating recombinantproteins produce sufficient quantities of proteins that retain theirrelevant biological activities and are of sufficient purity to optimizeactivity and assure assay reproducibility. Yeast two-hybrid and variantscreens, and mass spectrometry provide preferred methods for determiningprotein-protein interactions and elucidation of protein complexes. Incertain applications, when P5CR-interacting proteins are used in screensto identify small molecule modulators, the binding specificity of theinteracting protein to the P5CR protein may be assayed by various knownmethods such as substrate processing (e.g. ability of the candidateP5CR-specific binding agents to function as negative effectors inP5CR-expressing cells), binding equilibrium constants (usually at leastabout 10⁷ M⁻¹, preferably at least about 10⁸ M⁻¹, more preferably atleast about 10⁹ M⁻¹), and immunogenicity (e.g. ability to elicit P5CRspecific antibody in a heterologous host such as a mouse, rat, goat orrabbit). For enzymes and receptors, binding may be assayed by,respectively, substrate and ligand processing.

[0075] The screening assay may measure a candidate agent's ability tospecifically bind to or modulate activity of a P5CR polypeptide, afusion protein thereof, or to cells or membranes bearing the polypeptideor fusion protein. The P5CR polypeptide can be full length or a fragmentthereof that retains functional P5CR activity. The P5CR polypeptide maybe fused to another polypeptide, such as a peptide tag for detection oranchoring, or to another tag. The P5CR polypeptide is preferably humanP5CR, or is an ortholog or derivative thereof as described above. In apreferred embodiment, the screening assay detects candidate agent-basedmodulation of P5CR interaction with a binding target, such as anendogenous or exogenous protein or other substrate that hasP5CR-specific binding activity, and can be used to assess normal P5CRgene function.

[0076] Suitable assay formats that may be adapted to screen for P5CRmodulators are known in the art. Preferred screening assays are highthroughput or ultra high throughput and thus provide automated,cost-effective means of screening compound libraries for lead compounds(Fernandes P B, Curr Opin Chem Biol (1998) 2:597-603; Sundberg S A, CurrOpin Biotechnol 2000, 11:47-53). In one preferred embodiment, screeningassays uses fluorescence technologies, including fluorescencepolarization, time-resolved fluorescence, and fluorescence resonanceenergy transfer. These systems offer means to monitor protein-protein orDNA-protein interactions in which the intensity of the signal emittedfrom dye-labeled molecules depends upon their interactions with partnermolecules (e.g., Selvin P R, Nat Struct Biol (2000) 7:730-4; Fernandes PB, supra; Hertzberg R P and Pope A J, Curr Opin Chem Biol (2000)4:445-451).

[0077] A variety of suitable assay systems may be used to identifycandidate P5CR and p53 pathway modulators (e.g. U.S. Pat. No. 6,020,135(p53 modulation), U.S. Pat. Nos. 5,550,019 and 6,133,437 (apoptosisassays); and U.S. Pat. No. 6,114,132 (phosphatase and protease assays)).

[0078] Reductases are enzymes of oxidoreductase class that catalyzereactions in which metabolites are reduced. High throughput screeningassays for reductases may involve scintillation (Fernandes P B. (1998)Curr Opin Chem Biol 2:597-603; Delaporte E et al. (2001) J Biomol Screen6:225-231).

[0079] Apoptosis assays. Assays for apoptosis may be performed byterminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nickend labeling (TUNEL) assay. The TUNEL assay is used to measure nuclearDNA fragmentation characteristic of apoptosis (Lazebnik et al., 1994,Nature 371, 346), by following the incorporation of fluorescein-dUTP(Yonehara et al., 1989, J. Exp. Med. 169, 1747). Apoptosis may furtherbe assayed by acridine orange staining of tissue culture cells (Lucas,R., et al., 1998, Blood 15:4730-41). An apoptosis assay system maycomprise a cell that expresses a P5CR, and that optionally has defectivep53 function (e.g. p53 is over-expressed or under-expressed relative towild-type cells). A test agent can be added to the apoptosis assaysystem and changes in induction of apoptosis relative to controls whereno test agent is added, identify candidate p53 modulating agents. Insome embodiments of the invention, an apoptosis assay may be used as asecondary assay to test a candidate p53 modulating agents that isinitially identified using a cell-free assay system. An apoptosis assaymay also be used to test whether P5CR function plays a direct role inapoptosis. For example, an apoptosis assay may be performed on cellsthat over- or under-express P5CR relative to wild type cells.Differences in apoptotic response compared to wild type cells suggeststhat the P5CR plays a direct role in the apoptotic response. Apoptosisassays are described further in U.S. Pat. No. 6,133,437.

[0080] Cell proliferation and cell cycle assays. Cell proliferation maybe assayed via bromodeoxyuridine (BRDU) incorporation. This assayidentifies a cell population undergoing DNA synthesis by incorporationof BRDU into newly-synthesized DNA. Newly-synthesized DNA may then bedetected using an anti-BRDU antibody (Hoshino et al., 1986, Int. J.Cancer 38, 369; Campana et al., 1988, J. Immunol. Meth. 107, 79), or byother means.

[0081] Cell Proliferation may also be examined using [³H]-thymidineincorporation (Chen, J., 1996, Oncogene 13:1395-403; Jeoung, J., 1995,J. Biol. Chem. 270:18367-73). This assay allows for quantitativecharacterization of S-phase DNA syntheses. In this assay, cellssynthesizing DNA will incorporate [³H]-thymidine into newly synthesizedDNA. Incorporation can then be measured by standard techniques such asby counting of radioisotope in a scintillation counter (e.g., Beckman LS 3800 Liquid Scintillation Counter).

[0082] Cell proliferation may also be assayed by colony formation insoft agar (Sambrook et al., Molecular Cloning, Cold Spring Harbor(1989)). For example, cells transformed with P5CR are seeded in softagar plates, and colonies are measured and counted after two weeksincubation.

[0083] Involvement of a gene in the cell cycle may be assayed by flowcytometry (Gray J W et al. (1986) Int J Radiat Biol Relat Stud Phys ChemMed 49:237-55). Cells transfected with a P5CR may be stained withpropidium iodide and evaluated in a flow cytometer (available fromBecton Dickinson).

[0084] Accordingly, a cell proliferation or cell cycle assay system maycomprise a cell that expresses a P5CR, and that optionally has defectivep53 function (e.g. p53 is over-expressed or under-expressed relative towild-type cells). A test agent can be added to the assay system andchanges in cell proliferation or cell cycle relative to controls whereno test agent is added, identify candidate p53 modulating agents. Insome embodiments of the invention, the cell proliferation or cell cycleassay may be used as a secondary assay to test a candidate p53modulating agents that is initially identified using another assaysystem such as a cell-free kinase assay system. A cell proliferationassay may also be used to test whether P5CR function plays a direct rolein cell proliferation or cell cycle. For example, a cell proliferationor cell cycle assay may be performed on cells that over- orunder-express P5CR relative to wild type cells. Differences inproliferation or cell cycle compared to wild type cells suggests thatthe P5CR plays a direct role in cell proliferation or cell cycle.

[0085] Angiogenesis. Angiogenesis may be assayed using various humanendothelial cell systems, such as umbilical vein, coronary artery, ordermal cells. Suitable assays include Alamar Blue based assays(available from Biosource International) to measure proliferation;migration assays using fluorescent molecules, such as the use of BectonDickinson Falcon HTS FluoroBlock cell culture inserts to measuremigration of cells through membranes in presence or absence ofangiogenesis enhancer or suppressors; and tubule formation assays basedon the formation of tubular structures by endothelial cells on Matrigel®(Becton Dickinson). Accordingly, an angiogenesis assay system maycomprise a cell that expresses a P5CR, and that optionally has defectivep53 function (e.g. p53 is over-expressed or under-expressed relative towild-type cells). A test agent can be added to the angiogenesis assaysystem and changes in angiogenesis relative to controls where no testagent is added, identify candidate p53 modulating agents. In someembodiments of the invention, the angiogenesis assay may be used as asecondary assay to test a candidate p53 modulating agents that isinitially identified using another assay system. An angiogenesis assaymay also be used to test whether P5CR function plays a direct role incell proliferation. For example, an angiogenesis assay may be performedon cells that over- or under-express P5CR relative to wild type cells.Differences in angiogenesis compared to wild type cells suggests thatthe P5CR plays a direct role in angiogenesis.

[0086] Hypoxic induction. The alpha subunit of the transcription factor,hypoxia inducible factor-1 (HIF-1), is upregulated in tumor cellsfollowing exposure to hypoxia in vitro. Under hypoxic conditions, HIF-1stimulates the expression of genes known to be important in tumour cellsurvival, such as those encoding glyolytic enzymes and VEGF. Inductionof such genes by hypoxic conditions may be assayed by growing cellstransfected with P5CR in hypoxic conditions (such as with 0.1% O2, 5%CO2, and balance N2, generated in a Napco 7001 incubator (PrecisionScientific)) and normoxic conditions, followed by assessment of geneactivity or expression by Taqman®. For example, a hypoxic inductionassay system may comprise a cell that expresses a P5CR, and thatoptionally has a mutated p53 (e.g. p53 is over-expressed orunder-expressed relative to wild-type cells). A test agent can be addedto the hypoxic induction assay system and changes in hypoxic responserelative to controls where no test agent is added, identify candidatep53 modulating agents. In some embodiments of the invention, the hypoxicinduction assay may be used as a secondary assay to test a candidate p53modulating agents that is initially identified using another assaysystem. A hypoxic induction assay may also be used to test whether P5CRfunction plays a direct role in the hypoxic response. For example, ahypoxic induction assay may be performed on cells that over- orunder-express P5CR relative to wild type cells. Differences in hypoxicresponse compared to wild type cells suggests that the P5CR plays adirect role in hypoxic induction.

[0087] Cell adhesion. Cell adhesion assays measure adhesion of cells topurified adhesion proteins, or adhesion of cells to each other, inpresence or absence of candidate modulating agents. Cell-proteinadhesion assays measure the ability of agents to modulate the adhesionof cells to purified proteins. For example, recombinant proteins areproduced, diluted to 2.5 g/mL in PBS, and used to coat the wells of amicrotiter plate. The wells used for negative control are not coated.Coated wells are then washed, blocked with 1% BSA, and washed again.Compounds are diluted to 2× final test concentration and added to theblocked, coated wells. Cells are then added to the wells, and theunbound cells are washed off. Retained cells are labeled directly on theplate by adding a membrane-permeable fluorescent dye, such ascalcein-AM, and the signal is quantified in a fluorescent microplatereader.

[0088] Cell-cell adhesion assays measure the ability of agents tomodulate binding of cell adhesion proteins with their native ligands.These assays use cells that naturally or recombinantly express theadhesion protein of choice. In an exemplary assay, cells expressing thecell adhesion protein are plated in wells of a multiwell plate. Cellsexpressing the ligand are labeled with a membrane-permeable fluorescentdye, such as BCECF , and allowed to adhere to the monolayers in thepresence of candidate agents. Unbound cells are washed off, and boundcells are detected using a fluorescence plate reader.

[0089] High-throughput cell adhesion assays have also been described. Inone such assay, small molecule ligands and peptides are bound to thesurface of microscope slides using a microarray spotter, intact cellsare then contacted with the slides, and unbound cells are washed off. Inthis assay, not only the binding specificity of the peptides andmodulators against cell lines are determined, but also the functionalcell signaling of attached cells using immunofluorescence techniques insitu on the microchip is measured (Falsey J R et al., Bioconjug Chem.2001 May-Jun;12(3):346-53).

[0090] Primary Assays for Antibody Modulators

[0091] For antibody modulators, appropriate primary assays test is abinding assay that tests the antibody's affinity to and specificity forthe P5CR protein. Methods for testing antibody affinity and specificityare well known in the art (Harlow and Lane, 1988, 1999, supra). Theenzyme-linked immunosorbant assay (ELISA) is a preferred method fordetecting P5CR-specific antibodies; others include FACS assays,radioimmunoassays, and fluorescent assays.

[0092] Primary Assays for Nucleic Acid Modulators

[0093] For nucleic acid modulators, primary assays may test the abilityof the nucleic acid modulator to inhibit or enhance P5CR geneexpression, preferably mRNA expression. In general, expression analysiscomprises comparing P5CR expression in like populations of cells (e.g.,two pools of cells that endogenously or recombinantly express P5CR) inthe presence and absence of the nucleic acid modulator. Methods foranalyzing mRNA and protein expression are well known in the art. Forinstance, Northern blotting, slot blotting, ribonuclease protection,quantitative RT-PCR (e.g., using the TaqMan®, PE Applied Biosystems), ormicroarray analysis may be used to confirm that P5CR mRNA expression isreduced in cells treated with the nucleic acid modulator (e.g., CurrentProtocols in Molecular Biology (1994) Ausubel F M et al., eds., JohnWiley & Sons, Inc., chapter 4; Freeman W M et al., Biotechniques (1999)26:112-125; Kallioniemi O P, Ann Med 2001, 33:142-147; Blohm D H andGuiseppi-Elie, A Curr Opin Biotechnol 2001, 12:41-47). Proteinexpression may also be monitored. Proteins are most commonly detectedwith specific antibodies or antisera directed against either the P5CRprotein or specific peptides. A variety of means including Westernblotting, ELISA, or in situ detection, are available (Harlow E and LaneD, 1988 and 1999, supra).

[0094] Secondary Assays

[0095] Secondary assays may be used to further assess the activity ofP5CR-modulating agent identified by any of the above methods to confirmthat the modulating agent affects P5CR in a manner relevant to the p53pathway. As used herein, P5CR-modulating agents encompass candidateclinical compounds or other agents derived from previously identifiedmodulating agent. Secondary assays can also be used to test the activityof a modulating agent on a particular genetic or biochemical pathway orto test the specificity of the modulating agent's interaction with P5CR.

[0096] Secondary assays generally compare like populations of cells oranimals (e.g., two pools of cells or animals that endogenously orrecombinantly express P5CR) in the presence and absence of the candidatemodulator. In general, such assays test whether treatment of cells oranimals with a candidate P5CR-modulating agent results in changes in thep53 pathway in comparison to untreated (or mock- or placebo-treated)cells or animals. Certain assays use “sensitized genetic backgrounds”,which, as used herein, describe cells or animals engineered for alteredexpression of genes in the p53 or interacting pathways.

[0097] Cell-Based Assays

[0098] Cell based assays may use a variety of mammalian cell lines knownto have defective p53 function (e.g. SAOS-2 osteoblasts, H1299 lungcancer cells, C33A and HT3 cervical cancer cells, HT-29 and DLD-1 coloncancer cells, among others, available from American Type CultureCollection (ATCC), Manassas, Va.). Cell based assays may detectendogenous p53 pathway activity or may rely on recombinant expression ofp53 pathway components. Any of the aforementioned assays may be used inthis cell-based format. Candidate modulators are typically added to thecell media but may also be injected into cells or delivered by any otherefficacious means.

[0099] Animal Assays

[0100] A variety of non-human animal models of normal or defective p53pathway may be used to test candidate P5CR modulators. Models fordefective p53 pathway typically use genetically modified animals thathave been engineered to mis-express (e.g., over-express or lackexpression in) genes involved in the p53 pathway. Assays generallyrequire systemic delivery of the candidate modulators, such as by oraladministration, injection, etc.

[0101] In a preferred embodiment, p53 pathway activity is assessed bymonitoring neovascularization and angiogenesis. Animal models withdefective and normal p53 are used to test the candidate modulator'saffect on P5CR in Matrigel® assays. Matrigel® is an extract of basementmembrane proteins, and is composed primarily of laminin, collagen IV,and heparin sulfate proteoglycan. It is provided as a sterile liquid at4° C., but rapidly forms a solid gel at 37° C. Liquid Matrigel® is mixedwith various angiogenic agents, such as bFGF and VEGF, or with humantumor cells which over-express the P5CR. The mixture is then injectedsubcutaneously (SC) into female athymic nude mice (Taconic, Germantown,N.Y.) to support an intense vascular response. Mice with Matrigel®pellets may be dosed via oral (PO), intraperitoneal (IP), or intravenous(IV) routes with the candidate modulator. Mice are euthanized 5-12 dayspost-injection, and the Matrigel® pellet is harvested for hemoglobinanalysis (Sigma plasma hemoglobin kit). Hemoglobin content of the gel isfound to correlate the degree of neovascularization in the gel.

[0102] In another preferred embodiment, the effect of the candidatemodulator on P5CR is assessed via tumorigenicity assays. In one example,xenograft human tumors are implanted SC into female athymic mice, 6-7week old, as single cell suspensions either from a pre-existing tumor orfrom in vitro culture. The tumors which express the P5CR endogenouslyare injected in the flank, 1×10⁵ to 1×10⁷ cells per mouse in a volume of100 μL using a 27 gauge needle. Mice are then ear tagged and tumors aremeasured twice weekly. Candidate modulator treatment is initiated on theday the mean tumor weight reaches 100 mg. Candidate modulator isdelivered IV, SC, IP, or PO by bolus administration. Depending upon thepharmacokinetics of each unique candidate modulator, dosing can beperformed multiple times per day. The tumor weight is assessed bymeasuring perpendicular diameters with a caliper and calculated bymultiplying the measurements of diameters in two dimensions. At the endof the experiment, the excised tumors maybe utilized for biomarkeridentification or further analyses. For immunohistochemistry staining,xenograft tumors are fixed in 4% paraformaldehyde, 0.1M phosphate, pH7.2, for 6 hours at 4° C., immersed in 30% sucrose in PBS, and rapidlyfrozen in isopentane cooled with liquid nitrogen.

[0103] Diagnostic and Therapeutic Uses

[0104] Specific P5CR-modulating agents are useful in a variety ofdiagnostic and therapeutic applications where disease or diseaseprognosis is related to defects in the p53 pathway, such as angiogenic,apoptotic, or cell proliferation disorders. Accordingly, the inventionalso provides methods for modulating the p53 pathway in a cell,preferably a cell pre-determined to have defective p53 function,comprising the step of administering an agent to the cell thatspecifically modulates P5CR activity. Preferably, the modulating agentproduces a detectable phenotypic change in the cell indicating that thep53 function is restored, i.e., for example, the cell undergoes normalproliferation or progression through the cell cycle.

[0105] The discovery that P5CR is implicated in p53 pathway provides fora variety of methods that can be employed for the diagnostic andprognostic evaluation of diseases and disorders involving defects in thep53 pathway and for the identification of subjects having apredisposition to such diseases and disorders.

[0106] Various expression analysis methods can be used to diagnosewhether P5CR expression occurs in a particular sample, includingNorthern blotting, slot blotting, ribonuclease protection, quantitativeRT-PCR, and microarray analysis. (e.g., Current Protocols in MolecularBiology (1994) Ausubel F M et al., eds., John Wiley & Sons, Inc.,chapter 4; Freeman W M et al., Biotechniques (1999) 26:112-125;Kallioniemi O P, Ann Med 2001, 33:142-147; Blohm and Guiseppi-Elie, CurrOpin Biotechnol 2001, 12:41-47). Tissues having a disease or disorderimplicating defective p53 signaling that express a P5CR, are identifiedas amenable to treatment with a P5CR modulating agent. In a preferredapplication, the p53 defective tissue overexpresses a P5CR relative tonormal tissue. For example, a Northern blot analysis of mRNA from tumorand normal cell lines, or from tumor and matching normal tissue samplesfrom the same patient, using full or partial P5CR cDNA sequences asprobes, can determine whether particular tumors express or overexpressP5CR. Alternatively, the TaqMan® is used for quantitative RT-PCRanalysis of P5CR expression in cell lines, normal tissues and tumorsamples (PE Applied Biosystems).

[0107] Various other diagnostic methods may be performed, for example,utilizing reagents such as the P5CR oligonucleotides, and antibodiesdirected against a P5CR, as described above for: (1) the detection ofthe presence of P5CR gene mutations, or the detection of either over- orunder-expression of P5CR mRNA relative to the non-disorder state; (2)the detection of either an over- or an under-abundance of P5CR geneproduct relative to the non-disorder state; and (3) the detection ofperturbations or abnormalities in the signal transduction pathwaymediated by P5CR.

[0108] Thus, in a specific embodiment, the invention is drawn to amethod for diagnosing a disease in a patient, the method comprising: a)obtaining a biological sample from the patient; b) contacting the samplewith a probe for P5CR expression; c) comparing results from step (b)with a control; and d) determining whether step (c) indicates alikelihood of disease. Preferably, the disease is cancer, mostpreferably a cancer as shown in TABLE 1. The probe may be either DNA orprotein, including an antibody.

EXAMPLES

[0109] The following experimental section and examples are offered byway of illustration and not by way of limitation.

[0110] VIII. Drosophila p53 Screen

[0111] The Drosophila p53 gene was overexpressed specifically in thewing using the vestigial margin quadrant enhancer. Increasing quantitiesof Drosophila p53 (titrated using different strength transgenic insertsin 1 or 2 copies) caused deterioration of normal wing morphology frommild to strong, with phenotypes including disruption of pattern andpolarity of wing hairs, shortening and thickening of wing veins,progressive crumpling of the wing and appearance of dark “death”inclusions in wing blade. In a screen designed to identify enhancers andsuppressors of Drosophila p53, homozygous females carrying two copies ofp53 were crossed to 5663 males carrying random insertions of a piggyBactransposon (Fraser M et al., Virology (1985) 145:356-361). Progenycontaining insertions were compared to non-insertion-bearing siblingprogeny for enhancement or suppression of the p53 phenotypes. Sequenceinformation surrounding the piggyBac insertion site was used to identifythe modifier genes. Modifiers of the wing phenotype were identified asmembers of the p53 pathway. Drosophila p5cr was an enhancer of the wingphenotype. Human orthologs of the modifiers are referred to herein asP5CR.

[0112] VIII. P5CR Assay

[0113] In an assay based on fluorescence intensity, P5CR is quantifiedusing a homogeneous fluorescence HTS assay format, not requiring anywash steps. The assay is carried out in plates with any number of wells,such as 96, 384, 1536, or others.

[0114] In this assay, P5CR catalyzes the reduction of Pyrroline 5carboxylate to proline using NADPH. The reaction is allowed to proceedfor a period of time, and then stopped. Remaining NADPH is quantitatedvia a coupling reaction with resazurine and diaphorase to produce highlyfluorescent compound resorufin. As such, an inhibited P5CR reaction ischaracterized by a large resorufin signal. Conversely, an uninhibitedreaction produces a small resorufin signal.

[0115] Reaction conditions include 20 μM NADPH, and 100 μM P5C in 30 mMpotassium phosphate buffer (pH 7.0), in a total volume of up to 80 μl.Reactants are incubated for 1 hr to allow the reaction to proceed.Resazurine and diaphorase are then added at 20 μM and 50 mU,respectively. Increase in fluorescence intensity is then monitored on aplate reader, with excitation and emission intensities set to 540 and605 nm, respectively. Alternatively, reaction progress is monitoreddirectly by observing the consumption of NADPH either fluorimetrically(excitation/emission=340/360 nm) or spectrophotometrically at 340 nm.

[0116] VIII. High-Throughput in vitro Fluorescence Polarization Assay

[0117] Fluorescently-labeled P5CR peptide/substrate are added to eachwell of a 96-well microtiter plate, along with a test agent in a testbuffer (10 mM HEPES, 10 mM NaCl, 6 mM magnesium chloride, pH 7.6).Changes in fluorescence polarization, determined by using a FluoroliteFPM-2 Fluorescence Polarization Microtiter System (DynatechLaboratories, Inc), relative to control values indicates the testcompound is a candidate modifier of P5CR activity.

[0118] VIII. High-Throughput in vitro Binding Assay.

[0119]³³P-labeled P5CR peptide is added in an assay buffer (100 mM KCl,20 mM HEPES pH 7.6, 1 mM MgCl₂, 1% glycerol, 0.5% NP-40, 50 mMbeta-mercaptoethanol, 1 mg/ml BSA, cocktail of protease inhibitors)along with a test agent to the wells of a Neutralite-avidin coated assayplate and incubated at 25° C. for 1 hour. Biotinylated substrate is thenadded to each well and incubated for 1 hour. Reactions are stopped bywashing with PBS, and counted in a scintillation counter. Test agentsthat cause a difference in activity relative to control without testagent are identified as candidate p53 modulating agents.

[0120] VIII. Immunoprecipitations and Immunoblotting

[0121] For coprecipitation of transfected proteins, 3×10⁶ appropriaterecombinant cells containing the P5CR proteins are plated on 10-cmdishes and transfected on the following day with expression constructs.The total amount of DNA is kept constant in each transfection by addingempty vector. After 24 h, cells are collected, washed once withphosphate-buffered saline and lysed for 20 min on ice in 1 ml of lysisbuffer containing 50 mM Hepes, pH 7.9, 250 mM NaCl, 20mM-glycerophosphate, 1 mM sodium orthovanadate, 5 mM p-nitrophenylphosphate, 2 mM dithiothreitol, protease inhibitors (complete, RocheMolecular Biochemicals), and 1% Nonidet P-40. Cellular debris is removedby centrifugation twice at 15,000× g for 15 min. The cell lysate isincubated with 25 μl of M2 beads (Sigma) for 2 h at 4° C. with gentlerocking.

[0122] After extensive washing with lysis buffer, proteins bound to thebeads are solubilized by boiling in SDS sample buffer, fractionated bySDS-polyacrylamide gel electrophoresis, transferred to polyvinylidenedifluoride membrane and blotted with the indicated antibodies. Thereactive bands are visualized with horseradish peroxidase coupled to theappropriate secondary antibodies and the enhanced chemiluminescence(ECL) Western blotting detection system (Amersham Pharmacia Biotech).

[0123] VIII. Expression Analysis

[0124] All cell lines used in the following experiments are NCI(National Cancer Institute) lines, and are available from ATCC (AmericanType Culture Collection, Manassas, Va. 20110-2209). Normal and tumortissues were obtained from Impath, UC Davis, Clontech, Stratagene, andAmbion.

[0125] TaqMan analysis was used to assess expression levels of thedisclosed genes in various samples.

[0126] RNA was extracted from each tissue sample using Qiagen (Valencia,Calif.) RNeasy kits, following manufacturer's protocols, to a finalconcentration of 50 ng/μl. Single stranded cDNA was then synthesized byreverse transcribing the RNA samples using random hexamers and 500 ng oftotal RNA per reaction, following protocol 4304965 of Applied Biosystems(Foster City, Calif., http://www.appliedbiosystems.com/).

[0127] Primers for expression analysis using TaqMan assay (AppliedBiosystems, Foster City, Calif.) were prepared according to the TaqManprotocols, and the following criteria: a) primer pairs were designed tospan introns to eliminate genomic contamination, and b) each primer pairproduced only one product.

[0128] Taqman reactions were carried out following manufacturer'sprotocols, in 25 μl total volume for 96-well plates and 10 μl totalvolume for 384-well plates, using 300 nM primer and 250 nM probe, andapproximately 25 ng of cDNA. The standard curve for result analysis wasprepared using a universal pool of human cDNA samples, which is amixture of cDNAs from a wide variety of tissues so that the chance thata target will be present in appreciable amounts is good. The raw datawere normalized using 18S rRNA (universally expressed in all tissues andcells).

[0129] For each expression analysis, tumor tissue samples were comparedwith matched normal tissues from the same patient. A gene was consideredoverexpressed in a tumor when the level of expression of the gene was 2fold or higher in the tumor compared with its matched normal sample. Incases where normal tissue was not available, a universal pool of cDNAsamples was used instead. In these cases, a gene was consideredoverexpressed in a tumor sample when the difference of expression levelsbetween a tumor sample and the average of all normal samples from thesame tissue type was greater than 2 times the standard deviation of allnormal samples (i.e., Tumor−average(all normal samples)>2×STDEV(allnormal samples)).

[0130] Results are shown in Table 1. Data presented in bold indicatethat greater than 50% of tested tumor samples of the tissue typeindicated in row 1 exhibited over expression of the gene listed incolumn 1, relative to normal samples. Underlined data indicates thatbetween 25% to 49% of tested tumor samples exhibited over expression. Amodulator identified by an assay described herein can be furthervalidated for therapeutic effect by administration to a tumor in whichthe gene is overexpressed. A decrease in tumor growth confirmstherapeutic utility of the modulator. Prior to treating a patient withthe modulator, the likelihood that the patient will respond to treatmentcan be diagnosed by obtaining a tumor sample from the patient, andassaying for expression of the gene targeted by the modulator. Theexpression data for the gene(s) can also be used as a diagnostic markerfor disease progression. The assay can be performed by expressionanalysis as described above, by antibody directed to the gene target, orby any other available detection method. TABLE 1 NA kid- GI # breastcolon ney lung ovary 189497 P5CR1 0 3 12 30 0 0 7 4 7 (SEQ ID NO:1)

[0131] VIII. P5CR RNAi

[0132] RNAi experiments were carried out to knock down expression ofP5CR using small interfering RNAs (siRNA, Elbashir et al, supra). Thesequence of SEQ ID NO: 1 was used as a template for siRNAs. siRNAs(21mer, double stranded RNA oligos with 2 base 3′ overhangs) weretransfected into SW620 cells, a colon carcinoma cell line (availablefrom American Type Culture Collection (ATCC), Manassas, Va.) at 200 nMusing oligofectamine (Invitrogen) (day 1). Cells were incubated for 2days at 37 degrees, then split a second time (day 3) and re-transfectedthe next day (day 4). The experiment was ended on day 7, when cells wereharvested for protein extracts and used in western analysis.

[0133] P5CR protein was specifically knocked down as measured by westernanalysis using a mouse polyclonal antibody raised against P5CR. This wasin comparison with negative controls for mock transfection, and anon-specific siRNA (luciferase).

[0134] VIII. P5CR Immunohistochemistry

[0135] Immunohistochemistry was used to localize P5CR protein in humantissue sections according to known methods (Thomas Boenisch, ed. (2001)Handbook, Immunochemical Staining Methods, 3^(rd) Edition, DakoCorporation, Carpinteria, Calif., USA,http://www.dakousa.com/ihcbook/hbcontent.htm). P5CR was localized with apurified rabbit polyclonal antibody, and gave a punctate cytoplasmicstaining pattern. Weak staining for P5CR was seen in normal breast andsalivary gland. Screening of cancer tissues showed P5CR protein waspresent in breast, stomach, prostate, bladder, and salivary glandtumors.

1 14 1 1792 DNA Homo sapiens 1 ctccggacag catgagcgtg ggcttcatcggcgctggcca gctggctttt gccctggcca 60 agggcttcac agcagcaggc gtcttggctgcccacaagat aatggctagc tccccagaca 120 tggacctggc cacagtttct gctctcaggaagatgggggt gaagttgaca ccccacaaca 180 aggagacggt gcagcacagt gatgtgctcttcctggctgt gaagccacac atcatcccct 240 tcatcctgga tgaaataggc gccgacattgaggacagaca cattgtggtg tcctgcgcgg 300 ccggcgtcac catcagctcc attgagaagaagctgtcagc gtttcggcca gcccccaggg 360 tcatccgctg catgaccaac actccagtcgtggtgcggga gggggccacc gtgtatgcca 420 caggcacgca cgcccaggtg gaggacgggaggctcatgga gcagctgctg agcacggtgg 480 gcttctgcac ggaggtggaa gaggacctgattgatgccgt cacggggctc agtggcagcg 540 gccccgccta cgcattcaca gccctggatgccctggctga tgggggtgtg aagatgggac 600 ttccaaggcg cctggcagtc cgcctcggggcccaggccct cctgggggct gccaagatgc 660 tgctgcactc agaacagcac ccaggccagctcaaggacaa cgtcagctct cctggtgggg 720 ccaccatcca tgccttgcat gtgctggagagtgggggctt ccgctccctg ctcatcaacg 780 ctgtggaggc ctcctgcatc cgcacacgggagctgcagtc catggctgac caggagcagg 840 tgtcaccagc cgccatcaag aagaccatcctggacaaggt gaagctggac tcccctgcag 900 ggaccgctct gtcgccttct ggccacaccaagctgctccc ccgcagcctg gccccagcgg 960 gcaaggattg acacgtcctg cctgaccaccatcctgccac caccttctct tctcttgtca 1020 ctagggggac tagggggtcc ccaaagtggcccactttctg tggctctgat cagcgcaggg 1080 gccagccagg gacatagcca gggaggggccacatcacttc ccactggaaa tctctgtggt 1140 ctgcaagtgc ttcccagccc agaacaggggtggattcccc aacctcaacc tcctttcttc 1200 tctgctccca aaccatgtca ggaccaccttcctctagagc tcgggagccc ggagggtctt 1260 cacccactcc tactccagta tcagctggcacgggctcctt cctgagagca aaggtcaagg 1320 accccctctg tgaaggctca gcagaggtgggatcccacgc cccctcccgg cccctccctg 1380 ccctccattc agggagaaac ctctccttcccgtgtgagaa gggccagagg gtccaggcat 1440 cccaagtcca gcgtgaaggg ccacagcccctcttggctgc caagcacgca gatcccatgg 1500 acatttgggg aaagggctcc ttgggctgctggtgaacttc tgtggccacc acctcctgct 1560 cctgacctcc ctgggagggt gctatcagttctgtcctggc cctttcagtt ttataagttg 1620 gtttccagcc cccagtgtcc tgacttctgtctgccacatg aggagggagg ccctgcctgt 1680 gtgggagggt ggttactgtg ggtggaatagtggaggcctt caactgatta gacaaggccc 1740 gcccacatct tggagggcat ctgccttactgattaaaatg tcaatgtaat ct 1792 2 2331 DNA Homo sapiens 2 agcgcagcggcgtccgaggc aacaagatgg cagctgcgga gccgtctccg cggcgcgtgg 60 gcttcgtgggcgcgggccgc atggcggggg ccatcgcgca gggcctcatc agagcaggaa 120 aagtggaagctcagcacata ctggccagtg caccaacaga caggaaccta tgtcactttc 180 aagctctgggttgccggacc acgcactcca accaggaggt gctacagagc tgcctgctcg 240 tcatctttgccaccaagcct catgtgctgc cagctgtcct ggcagaggtg gctcctgtgg 300 tcaccactgaacacatcttg gtgtccgtgg ctgctggggt gtctctgagc accctggagg 360 agctgctgcccccaaacaca cgggtgctgc gggtcttgcc caacctgccc tgtgtggtcc 420 aggaaggggccatagtgatg gcgcggggcc gccacgtggg gagcagcgag accaagctcc 480 tgcagcatctgctggaggcc tgtgggcggt gtgaggaggt gcctgaagcc tacgtcgaca 540 tccacactggcctcagtggc agtggcgtgg ccttcgtgtg tgcattctcc gaggccctgg 600 ctgaaggagccgtcaagatg ggcatgccca gcagcctggc ccaccgcatc gctgcccaga 660 ccctgctggggacggccaag atgctgctgc acgagggcca acacccagcc cagctgcgct 720 cagacgtgtgcaccccgggt ggcaccacca tctatggact ccacgccctg gagcagggcg 780 ggctgcgagcagccaccatg agcgccgtgg aggctgccac ctgccgggcc aaggagctca 840 gcagaaagtaggctgggctc tggccatcct ttcctgcctc tgtgcccctg cctctccctg 900 tgtcccttcccctgaggact gcggctccct ccctcctgca tgagggtctc ctactgctcc 960 ttctccccttgcacagggaa atgcaggggg caggacttgg gaggttccag caggcggggg 1020 agccccgaccagtggggaca ctcctccctc cccagtgagc agaaggcacc gtggtggtgg 1080 ctctgccccttgctgcagtg agcccacctt gctgcaacat tggttctgag gggcccaaga 1140 gatggcgtcttggtcatttg cccgcatggt tgggcagttg gttgaggcca tgaacagaac 1200 ttacggtaacaggcacggct ggcccaatgc ctggtctgga gctggagctt gcctttggct 1260 ttccaggtggctccgtgcag ctacagccag gccggctgcc tcatctcagc tctagggggc 1320 acgagccatatggggtctgc acaagagacc ctctcccctg cagtaaagcc aggggccctg 1380 gcctgatggggcccccatgg ggagctggag cctgccctgc agcctggaga agagggtggc 1440 tgtggtgggcgtgctcatcc cctgctaagg agcaggagct gctgggccag gtctgcggca 1500 gtgctggggtggcaccaggt gggcagtggt aggtggggtg gcttgaggtc tgggagggtg 1560 gccctggccagccaggacac atgcagaccc ctggcttagt ctggatacag gctccctctt 1620 tcctcccaatcctaagctcc tgacaagtgg ccaggtggct ctgggccctc ctgccccgtg 1680 cctaggtcaggggtcctgga ataccccgta gctctggcac caccacactg gcctctgatg 1740 gcaagacttggcccctccac ctgtccctaa cggacggcag gtcaggaaag ccaggactca 1800 ggggagaagcaaacccccag gattgaaggc tagggttcta gggcctttgg gtggggaggg 1860 cccgggccggacagcctcag ctccgtcccc tgccccacaa gattcacctg ggcctccagt 1920 cccacgctggccccaactgc tgcagctctc ggcttccgcc caacagcctc tggaggtgag 1980 gcgggagcatgccctcagcg aggctgggcg gcgggtcctg ctgtgccatc tccctgtgcg 2040 cctgagcagatcaatccacc agtgcaaaac agggctaacg gcacctgcag gacagcagca 2100 cgctccatccctcatgctca gctgcctctg cggccacgga cttctgccct tcatctgctc 2160 tctcttactctcctgagcct agcccgtccg taagctccct cccctgcctg gttcccaggg 2220 caggctgactcagttgactg cttggtccaa gcctggccct ggcacttgtc agggtcagcc 2280 taaggagatgggaataaaga ggccagagag caccaagtga gctcatgttt c 2331 3 1848 DNA Homosapiens 3 ggcacgaggg ccatctgtgg gggctttggg ccaggggtct ccggacagcatgagcgtggg 60 cttcatcggc gctggccagc tggcttttgc cctggccaag ggcttcacagcagcaggcgt 120 cttggctgcc cacaagataa tggctagctc cccagacatg gacctggccacagtttctgc 180 tctcaggaag atgggggtga agttgacacc ccacaacaag gagacggtgcagcacagtga 240 tgtgctcttc ctggctgtga agccacacat catccccttc atcctggatgaaataggcgc 300 cgacattgag gacagacaca ttgtggtgtc ctgcgcggcc ggcgtcaccatcagctccat 360 tgagaagaag ctgtcagcgt ttcggccagc ccccagggtc atccgctgcatgaccaacac 420 tccagtcgtg gtgcgggagg gggccaccgt gtatgccaca ggcacgcacgcccaggtgga 480 ggacgggagg ctcatggagc agctgctgag cagcgtgggc ttctgcacggaggtggaaga 540 ggacctgatt gatgccgtca cggggctcag tggcagcggc cccgcctacgcattcacagc 600 cctggatgcc ctggctgatg ggggcgtgaa gatgggactt ccaaggcgcctggcagtccg 660 cctcggggcc caggccctcc tgggggctgc caagatgctg ctgcactcagaacagcaccc 720 aggccagctc aaggacaacg tcagctctcc tggtggggcc accatccatgccttgcatgt 780 gctggagagt gggggcttcc gctccctgct catcaacgct gtggaggcctcctgcatccg 840 cacacgggag ctgcagtcca tggctgacca ggagcaggtg tcaccagccgccatcaagaa 900 gaccatcctg gacaaggtga agctggactc ccctgcaggg accgctctgtcgccttctgg 960 ccacaccaag ctgctccccc gcagcctggc cccagcgggc aaggattgacacgtcctgcc 1020 tgaccaccat cctgccacca ccttctcttc tcttgtcact agggggactagggggtcccc 1080 aaagtggccc actttctgtg gctctgatca gcgcaggggc cagccagggacatagccagg 1140 gaggggccac atcacttccc actggaaatc tctgtggtct gcaagtgcttcccagcccag 1200 aacaggggtg gattccccaa cctcaacctc ctttcttctc tgctcccaaaccatgtcagg 1260 accaccttcc tctagagctc gggagcccgg agggtcttca cccactcctactccagtatc 1320 agctggcacg ggctccttcc tgagagcaaa ggtcaaggac cccctctgtgaaggctcagc 1380 agaggtggga tcccacgccc cctcccggcc cctccctgcc ctccattcagggagaaacct 1440 ctccttcccg tgtgagaagg gccagagggt ccaggcatcc caagtccagcgtgaagggcc 1500 acagcccctc ttggctgcca agcacgcaga tcccatggac atttggggaaagggctcctt 1560 gggctgctgg tgaacttctg tggccaccac ctcctgctcc tgacctccctgggagggtgc 1620 tatcagttct gtcctggccc tttcagtttt ataagttggt ttccagcccccagtgtcctg 1680 acttctgtct gccacatgag gagggaggcc ctgcctgtgt gggagggtggttactgtggg 1740 tggaatagtg gaggccttca actgattaga caaggcccgc ccacatcttggagggcatct 1800 gccttactga ttaaaatgtc aatgtaatct aaaaaaaaaa aaaaaaaa1848 4 1028 DNA Homo sapiens misc_feature (999)..(999) “n”is A, C, G, orT 4 acccgcgcac gagccggtgc catctgtggg ggctttgggc caggggtctc cggacagcat 60gagcgtgggc ttcatcggcg ctggccagct ggcttttgcc ctggccaagg gcttcacagc 120agcaggcgtc ttggctgccc acaagataat ggctagctcc ccagacatgg acctggccac 180agtttctgct ctcaggaaga tgggggtgaa gttgacaccc cacaacaagg agacggtgca 240gcacagtgat gtgctcttcc tggctgtgaa gccacacatc atccccttca tcctggatga 300aataggcgcc gacattgagg acagacacat tgtggtgtcc tgcgcggccg gcgtcaccat 360cagctccatt gagaagaagc tgtcagcgtt tcggccagcc cccagggtca tccgctgcat 420gaccaacact ccagtcgtgg tgcgggaggg ggccaccgtg tatgccacag gcacgcacgc 480ccaggtggag gacgggaggc tcatggagca gctgctgagc agcgtgggct tctgcacgga 540ggtggaagag gacctgattg atgccgtcac ggggctcagt ggcagcggcc ccgcctacgc 600attcacagcc ctggatgccc tggctgatgg gggtgtgaag atgggacttc caaggcgcct 660ggcagtccgc ctcggggccc aggccctcct gggggctgcc aagatgctgc tgcactcaga 720acagcaccca ggccagctca aggacaacgt cagctctcct ggtgggggca ccatccatgc 780cttgcatgtg ctggagaagt gggggcttcc gctccctgct catcaacgct gtggaaggcc 840tcctgcatcc gcacaccggg agctgcagtc ccatggcctg accaagaagc aggtgttcac 900cagccggcat ccagaagaac atccctggga caaggtggaa actgggactc cccctgcaag 960gggaccggct cctgtcgcct ttcgggccca caaccaagnc tggcttcccc cgccagaccg 1020tgggcccc 1028 5 1715 DNA Homo sapiens 5 agtcatttct tctgcggaac ctccacccctccagtgggtg cgggacccta ggcagcaggc 60 caggcaccgc cgctctgact gggggccccgaagcagttaa cgggccggac agcaaagtgg 120 aaagttagac cagctgggac caggggaggtgggcacccgg ctgcgggagg agcggccagg 180 ctggcactgc ccccctaact tgctctcgatcctgccggtc tcgtcccgca gagccggtgc 240 catctgtggg ggctttgggc caggggtctccggacagcat gagcgtgggc ttcatcggcg 300 ctggccagct ggcttttgcc ctggccaaggctttcacagc agcaggcgtc ttggctgccc 360 acaagataat ggctagctcc ccagacatggacctggccac agtttctgct ctcaggaaga 420 tgggggtgaa gttgacaccc cacaacaaggagacggtgca gcacagtgat gtgctcttcc 480 tggctgtgaa gccacacatc atccccttcatcctggatga aataggcgcc gacattgagg 540 acagacacat tgtggtgtcc tgcgcggccggcgtcaccat cagctccatt gagaagaagc 600 tgtcagcgtt tcggccagcc cccagggtcatccgctgcat gaccaacact ccagtcgtgg 660 tgcgggaggg ggccaccgtg tatgccacaggacgcacgcc caggtggagg acgggaggct 720 catggagcag ctgctgagca gcgtgggcttctgcacggag gtggaagagg acctgattga 780 tgccgtcacg gggctcagtg gcagcggccccgcctacgca ttcacagccc tggatgccct 840 ggctgatggg ggtgtgaaga tgggacttccaaggcgcctg gcagtccgcc tcggggccca 900 ggccctcctg ggggctgcca agatgctgctgcactcagaa cagcacccag gccagctcaa 960 ggacaacgtc agctctcctg gtggggccaccatccatgcc ttgcatgtgc tggagagtgg 1020 gggcttccgc tccctgctca tcaacgctgtggaggcctcc tgcatccgca cacgggagct 1080 gcagtccatg gctgaccagg agcaggtgtcaccagccgcc atcaagaaga ccatcctgga 1140 caaggaccac cttcctctag agctcgggagcccggagggt cttcacccac tcctactcca 1200 gtatcagctg gcacgggctc cttcctgagagcaaaggtca aggaccccct ctgtgaaggc 1260 tcagcagagg tgggatccca cgccccctcccggcccctcc ctgccctcca ttcagggaga 1320 aacctctcct tcccgtgtga gaagggccagagggtccagg catcccaagt ccagcgtgaa 1380 gggccacagc ccctcttggc tgccaagcacgcagatccca tggacatttg gggaaagggc 1440 tccttgggct gctggtgaac ttctgtggccaccacctcct gctcctgacc tccctgggag 1500 ggtgctatca gttctgtcct ggccctttcagttttataag ttggtttcca gcccccagtg 1560 tcctgacttc tgtctgccac atgaggagggaggccctgcc tgtgtgggag ggtggttact 1620 gtgggtggaa tagtggaggc cttcaactgattagacaagg cccgcccaca tcttggaggg 1680 catctgcctt actgattaaa atgtcaatgtaatct 1715 6 860 DNA Homo sapiens 6 ggcgtccgag gcaacaagat ggcagctgcggagccgtctc cgcggcgcgt gggcttcgtg 60 ggcgcgggcc gcatggcggg ggccatcgcgcagggcctca tcagagcagg aaaagtggaa 120 gctcagcaca tactggccag tgcaccaacagacaggaacc tatgtcactt tcaagctctg 180 ggttgccgga ccacgcactc caaccaggaggtgctgcaga gctgcctgct cgtcatcttt 240 gccaccaagc ctcatgtgct gccagctgtcctggcagagg tggctcctgt ggtcaccact 300 gaacacatct tggtgtccgt ggctgctggggtgtctctga gcaccctgga ggagctgctg 360 cccccaaaca cacgggtgct gcgggtcttgcccaacctgc cctgtgtggt ccaggaaggg 420 gccatagtga tggcgcgggg ccgccacgtggggagcagcg agaccaagct cctgcagcat 480 ctgctggagg cctgtgggcg gtgtgaggaggtgcctgaag cctacgtcga catccacact 540 ggcctcagtg gcagtggcgt ggccttcgtgtgtgcattct ccgaggccct ggctgaagga 600 gccgtcaaga tgggcatgcc cagcagcctggcccaccgca tcgctgccca gaccctgctg 660 gggacggcca agatgctgct gcacgagggccaacacccag cccagctgcg ctcagacgtg 720 tgcaccccgg gtggcaccac catctatggactccacgccc tggagcaggg cgggctgcga 780 gcagccacca tgagcgccgt ggaggctgccacctgccggg ccaaggagct cagcagaaag 840 taggctgggc tctggccatc 860 7 1178DNA Homo sapiens 7 cggcctgtgg atgggcggtg agcgcagcgg cgtccgaggcaacaagatgg cagctgcgga 60 gccgtctccg cggcgcgtgg gcttcgtggg cgcgggccgcatggcggggg ccatcgcgca 120 gggcctcatc agagcaggaa aagtggaagc tcagcacatactggccagtg caccaacaga 180 caggaaccta tgtcactttc aagctctggg ttgccggaccacgcactcca accaggaggt 240 gctgcagagc tgcctgctcg tcatctttgc caccaagcctcatgtgctgc cagctgtcct 300 ggcagaggtg gctcctgtgg tcaccactga acacatcttggtgtccgtgg ctgctggggt 360 gtctctgagc accctggagg agctgctgcc cccaaacacacgggtgctgc gggtcttgcc 420 caacctgccc tgtgtggtcc aggaaggggc catagtgatggcgcggggcc gccacgtggg 480 gagcagcgag accaacctcc tgcagcatct gctggaggcctgtgggcggt gtgaggaggt 540 gcctgaagcc tacgtcgaca tccacactgg cctcagtggcagtggcgtgg ccttcgtgtg 600 tgcattctcc gaggccctgg ctgaaggagc cgtcaagatgggcatgccca gcagcctggc 660 ccaccgcatc gctgcccaga ccctgctggg gacggccaagatgctgctgc acgagggcca 720 acacccagcc cagctgcgct cagacgtgtg caccccgggtggcaccacca tctatggact 780 ccacgccctg gagcagggcg ggctgcgagc agccaccatgagcgccgtgg aggctgccac 840 ctgccgggcc aaggagctca gcagaaagta ggctgggctctggccatcct ttcctgcctc 900 tgtgcccctg cctctccctg tgtcccttcc cctgaggactgcggctccct ccctcctgca 960 tgagggtctc ctactgctcc ttctcccctt gcacagggaaatgcaggggg caggacttgg 1020 gaggttccag caggcggggg agccccgacc agtggggacactcctccctc cccagtgagc 1080 agaaggcacc gtggtggtgg ctctgcccct tgctgcagtgagcccacctt gctgcaacat 1140 tggttctgag gggcccaaga aaaaaaaaaa aaaaaaaa1178 8 1708 DNA Homo sapiens 8 gaatagggtt gcaccatccc agaagctgctgttagctcgc cggtcctcgg cacgccgccc 60 gttcgcccct gcgctgtccg cccttcccctagcgttactt ccggtccctc gctgaggggg 120 ttcgtgcggc tcccaggagg cgtgaaccgcggaccatgag cgtgggcttc atcggggccg 180 gccagctggc ctatgctctg gcgcggggcttcacggccgc aggcatcctg tcggctcaca 240 agataatagc cagctcccca gaaatgaacctgcccacggt gtccgcgctc aggaagatgg 300 gtgtgaacct gacacgcagc aacaaggagacggtgaagca cagcgacgtc ctgtttctgg 360 ctgtgaagcc acatatcatc cccttcatcctggatgagat tggggccgac gtgcaagcca 420 gacacatcgt ggtctcctgt gcggctggtgtcaccatcag ctctgtggag aagaagctga 480 tggcattcca gccagccccc aaagtgattcgctgcatgac caacacacct gtggtagtgc 540 aggaaggcgc tacagtgtac gccacgggcacccatgccct ggtggaggat gggcagctcc 600 tggagcagct catgagcagc gtgggcttctgcactgaggt ggaagaggac ctcatcgatg 660 ccgtcacggg gctcagtggc agcgggcctgcctatgcatt catggctctg gacgcattgg 720 ctgatggtgg ggtgaagatg ggtttgccacggcgcctggc aatccaactc ggggcccagg 780 ctttgctggg agctgccaag atgctgctggactcggagca gcatccatgc cagcttaagg 840 acaatgtctg ctcccctggg ggagccaccatccacgccct gcactttcta gagagtgggg 900 gcttccgctc tctgctcatc aatgcagttgaggcctcctg tatccgaaca cgagagctac 960 agtccatggc cgaccaagaa aagatctccccagctgccct taagaagacc ctcttagaca 1020 gagtgaagct ggaatccccc acagtctccacactgacccc ctccagccca gggaagctcc 1080 tcacaagaag cctggccctg ggaggcaagaaggactaagg cagcatctgt cccctctgtg 1140 attcagagcc cttagttgag agcccctgccgcccctgcca cccccctgcc ccgctcccac 1200 cattgcccct cctcagctgt gcaaggagaaagcatgctta ggaagttttc aggtccttgt 1260 gataaaacct ccttaaatct gttcagaccaagcaatgcga gcttcctctc ctgtcccatg 1320 ttggaagttg ctctgaaggg gtggtagatgctggaagcca gacacaaccc tgcgtacgct 1380 gctcagttgg tggagactgg ggctgggactggagtcagcc cagctgggag gaggggctgg 1440 ggaggatctg cagctgaagc ccgaggcagggttggtgtga tgccaaggca aagtggtgag 1500 gagaaaacag gaaacgggct ttctctgaattggtaaatgg gaaagaagtg agcaacttaa 1560 gattgtcaca attaatcaca agtgtacaggattagactgg gtttatattt aactcttgct 1620 tcataggtgt accatttaaa gagtgttatttaatgctaag tttaactgct ttaataaagt 1680 ttatttttaa ataaaaaaaa aaaaaaaa1708 9 999 DNA Homo sapiens 9 gcggctccca ggaggcgtga accgcggaccatgagcgtgg gcttcatcgg ggccggccag 60 ctggctatgc tctggcgcgg ggcttcacggccgcagattc ctgtcggctc acaagataat 120 agccagctcc ccagaaatga acctgcccacggtgtccgcg ctcaggaaga tgggtgtgaa 180 cctgacacgc agcaacaagg agacggtgaagcacagcgac gtcctgtttc tggctgtgaa 240 gcacatatca tccccttcat cctggttgagattggggccg acgtgcaagc cagacacatc 300 gtggtctcct gtgcggctgg tgttaccatcagctctgtgg agaagaagct gatggaattc 360 cagccagccc ccaaagtgat tcgctgcatgaccaacacac ctgtggtagt gcaggaaggc 420 gctacagtgt acgccacggg cacccatgccctggtggagg atgggcagct cctggagcag 480 ctcatgagca gcgtgggctt ctgcactgaggtggaagagg acctcatcga tgccgtcacg 540 gggctcagtg gcagcaaacc tgcctatgcattcatggctc tggacgcatt ggctgatggt 600 ggggtgaaga tgggtttgcc acggcgcctggcaatccaac tcggggccca ggctttgctg 660 ggagctgcca agatgctgct ggactcggagcagcatccat gccagcttaa ggacaatgtc 720 tgctcccctg ggggagccac catccacgccctgcactttc tagagagtgg gggcttccgc 780 tctctgctca tcaatgcagt tgaggcctcctgtatccgaa cacgagagct acagtccatg 840 gccgaccaag aaaagatctc cccagctgcccttaagaaga ccctcttaga cagagtgaag 900 ctggaatccc ccacagtctc cacactgaccccctccagcc cagggaagct cctcacaaga 960 agcctggccc tgggaggcaa gaaggactaaggcagcatc 999 10 319 PRT Homo sapiens 10 Met Ser Val Gly Phe Ile Gly AlaGly Gln Leu Ala Phe Ala Leu Ala 1 5 10 15 Lys Gly Phe Thr Ala Ala GlyVal Leu Ala Ala His Lys Ile Met Ala 20 25 30 Ser Ser Pro Asp Met Asp LeuAla Thr Val Ser Ala Leu Arg Lys Met 35 40 45 Gly Val Lys Leu Thr Pro HisAsn Lys Glu Thr Val Gln His Ser Asp 50 55 60 Val Leu Phe Leu Ala Val LysPro His Ile Ile Pro Phe Ile Leu Asp 65 70 75 80 Glu Ile Gly Ala Asp IleGlu Asp Arg His Ile Val Val Ser Cys Ala 85 90 95 Ala Gly Val Thr Ile SerSer Ile Glu Lys Lys Leu Ser Ala Phe Arg 100 105 110 Pro Ala Pro Arg ValIle Arg Cys Met Thr Asn Thr Pro Val Val Val 115 120 125 Arg Glu Gly AlaThr Val Tyr Ala Thr Gly Thr His Ala Gln Val Glu 130 135 140 Asp Gly ArgLeu Met Glu Gln Leu Leu Ser Thr Val Gly Phe Cys Thr 145 150 155 160 GluVal Glu Glu Asp Leu Ile Asp Ala Val Thr Gly Leu Ser Gly Ser 165 170 175Gly Pro Ala Tyr Ala Phe Thr Ala Leu Asp Ala Leu Ala Asp Gly Gly 180 185190 Val Lys Met Gly Leu Pro Arg Arg Leu Ala Val Arg Leu Gly Ala Gln 195200 205 Ala Leu Leu Gly Ala Ala Lys Met Leu Leu His Ser Glu Gln His Pro210 215 220 Gly Gln Leu Lys Asp Asn Val Ser Ser Pro Gly Gly Ala Thr IleHis 225 230 235 240 Ala Leu His Val Leu Glu Ser Gly Gly Phe Arg Ser LeuLeu Ile Asn 245 250 255 Ala Val Glu Ala Ser Cys Ile Arg Thr Arg Glu LeuGln Ser Met Ala 260 265 270 Asp Gln Glu Gln Val Ser Pro Ala Ala Ile LysLys Thr Ile Leu Asp 275 280 285 Lys Val Lys Leu Asp Ser Pro Ala Gly ThrAla Leu Ser Pro Ser Gly 290 295 300 His Thr Lys Leu Leu Pro Arg Ser LeuAla Pro Ala Gly Lys Asp 305 310 315 11 274 PRT Homo sapiens 11 Met AlaAla Ala Glu Pro Ser Pro Arg Arg Val Gly Phe Val Gly Ala 1 5 10 15 GlyArg Met Ala Gly Ala Ile Ala Gln Gly Leu Ile Arg Ala Gly Lys 20 25 30 ValGlu Ala Gln His Ile Leu Ala Ser Ala Pro Thr Asp Arg Asn Leu 35 40 45 CysHis Phe Gln Ala Leu Gly Cys Arg Thr Thr His Ser Asn Gln Glu 50 55 60 ValLeu Gln Ser Cys Leu Leu Val Ile Phe Ala Thr Lys Pro His Val 65 70 75 80Leu Pro Ala Val Leu Ala Glu Val Ala Pro Val Val Thr Thr Glu His 85 90 95Ile Leu Val Ser Val Ala Ala Gly Val Ser Leu Ser Thr Leu Glu Glu 100 105110 Leu Leu Pro Pro Asn Thr Arg Val Leu Arg Val Leu Pro Asn Leu Pro 115120 125 Cys Val Val Gln Glu Gly Ala Ile Val Met Ala Arg Gly Arg His Val130 135 140 Gly Ser Ser Glu Thr Lys Leu Leu Gln His Leu Leu Glu Ala CysGly 145 150 155 160 Arg Cys Glu Glu Val Pro Glu Ala Tyr Val Asp Ile HisThr Gly Leu 165 170 175 Ser Gly Ser Gly Val Ala Phe Val Cys Ala Phe SerGlu Ala Leu Ala 180 185 190 Glu Gly Ala Val Lys Met Gly Met Pro Ser SerLeu Ala His Arg Ile 195 200 205 Ala Ala Gln Thr Leu Leu Gly Thr Ala LysMet Leu Leu His Glu Gly 210 215 220 Gln His Pro Ala Gln Leu Arg Ser AspVal Cys Thr Pro Gly Gly Thr 225 230 235 240 Thr Ile Tyr Gly Leu His AlaLeu Glu Gln Gly Gly Leu Arg Ala Ala 245 250 255 Thr Met Ser Ala Val GluAla Ala Thr Cys Arg Ala Lys Glu Leu Ser 260 265 270 Arg Lys 12 319 PRTHomo sapiens 12 Met Ser Val Gly Phe Ile Gly Ala Gly Gln Leu Ala Phe AlaLeu Ala 1 5 10 15 Lys Gly Phe Thr Ala Ala Gly Val Leu Ala Ala His LysIle Met Ala 20 25 30 Ser Ser Pro Asp Met Asp Leu Ala Thr Val Ser Ala LeuArg Lys Met 35 40 45 Gly Val Lys Leu Thr Pro His Asn Lys Glu Thr Val GlnHis Ser Asp 50 55 60 Val Leu Phe Leu Ala Val Lys Pro His Ile Ile Pro PheIle Leu Asp 65 70 75 80 Glu Ile Gly Ala Asp Ile Glu Asp Arg His Ile ValVal Ser Cys Ala 85 90 95 Ala Gly Val Thr Ile Ser Ser Ile Glu Lys Lys LeuSer Ala Phe Arg 100 105 110 Pro Ala Pro Arg Val Ile Arg Cys Met Thr AsnThr Pro Val Val Val 115 120 125 Arg Glu Gly Ala Thr Val Tyr Ala Thr GlyThr His Ala Gln Val Glu 130 135 140 Asp Gly Arg Leu Met Glu Gln Leu LeuSer Thr Val Gly Phe Cys Thr 145 150 155 160 Glu Val Glu Glu Asp Leu IleAsp Ala Val Thr Gly Leu Ser Gly Ser 165 170 175 Gly Pro Ala Tyr Ala PheThr Ala Leu Asp Ala Leu Ala Asp Gly Gly 180 185 190 Val Lys Met Gly LeuPro Arg Arg Leu Ala Val Arg Leu Gly Ala Gln 195 200 205 Ala Leu Leu GlyAla Ala Lys Met Leu Leu His Ser Glu Gln His Pro 210 215 220 Gly Gln LeuLys Asp Asn Val Ser Ser Pro Gly Gly Ala Thr Ile His 225 230 235 240 AlaLeu His Val Leu Glu Ser Gly Gly Phe Arg Ser Leu Leu Ile Asn 245 250 255Ala Val Glu Ala Ser Cys Ile Arg Thr Arg Glu Leu Gln Ser Met Ala 260 265270 Asp Gln Glu Gln Val Ser Pro Ala Ala Ile Lys Lys Thr Ile Leu Asp 275280 285 Lys Val Lys Leu Asp Ser Pro Ala Gly Thr Ala Leu Ser Pro Ser Gly290 295 300 His Thr Lys Leu Leu Pro Arg Ser Leu Ala Pro Ala Gly Lys Asp305 310 315 13 274 PRT Homo sapiens 13 Met Ala Ala Ala Glu Pro Ser ProArg Arg Val Gly Phe Val Gly Ala 1 5 10 15 Gly Arg Met Ala Gly Ala IleAla Gln Gly Leu Ile Arg Ala Gly Lys 20 25 30 Val Glu Ala Gln His Ile LeuAla Ser Ala Pro Thr Asp Arg Asn Leu 35 40 45 Cys His Phe Gln Ala Leu GlyCys Arg Thr Thr His Ser Asn Gln Glu 50 55 60 Val Leu Gln Ser Cys Leu LeuVal Ile Phe Ala Thr Lys Pro His Val 65 70 75 80 Leu Pro Ala Val Leu AlaGlu Val Ala Pro Val Val Thr Thr Glu His 85 90 95 Ile Leu Val Ser Val AlaAla Gly Val Ser Leu Ser Thr Leu Glu Glu 100 105 110 Leu Leu Pro Pro AsnThr Arg Val Leu Arg Val Leu Pro Asn Leu Pro 115 120 125 Cys Val Val GlnGlu Gly Ala Ile Val Met Ala Arg Gly Arg His Val 130 135 140 Gly Ser SerGlu Thr Lys Leu Leu Gln His Leu Leu Glu Ala Cys Gly 145 150 155 160 ArgCys Glu Glu Val Pro Glu Ala Tyr Val Asp Ile His Thr Gly Leu 165 170 175Ser Gly Ser Gly Val Ala Phe Val Cys Ala Phe Ser Glu Ala Leu Ala 180 185190 Glu Gly Ala Val Lys Met Gly Met Pro Ser Ser Leu Ala His Arg Ile 195200 205 Ala Ala Gln Thr Leu Leu Gly Thr Ala Lys Met Leu Leu His Glu Gly210 215 220 Gln His Pro Ala Gln Leu Arg Ser Asp Val Cys Thr Pro Gly GlyThr 225 230 235 240 Thr Ile Tyr Gly Leu His Ala Leu Glu Gln Gly Gly LeuArg Ala Ala 245 250 255 Thr Met Ser Ala Val Glu Ala Ala Thr Cys Arg AlaLys Glu Leu Ser 260 265 270 Arg Lys 14 319 PRT Homo sapiens 14 Met SerVal Gly Phe Ile Gly Ala Gly Gln Leu Ala Met Leu Trp Arg 1 5 10 15 GlyAla Ser Arg Pro Gln Ile Pro Val Gly Ser Gln Asp Asn Ser Gln 20 25 30 LeuPro Arg Asn Glu Pro Ala His Gly Val Arg Ala Gln Glu Asp Gly 35 40 45 CysGlu Pro Asp Thr Gln Gln Gln Gly Asp Gly Glu Ala Gln Arg Arg 50 55 60 ProVal Ser Gly Cys Glu Ala His Ile Ile Pro Phe Ile Leu Val Glu 65 70 75 80Ile Gly Ala Asp Val Gln Ala Arg His Ile Val Val Ser Cys Ala Ala 85 90 95Gly Val Thr Ile Ser Ser Val Glu Lys Lys Leu Met Glu Phe Gln Pro 100 105110 Ala Pro Lys Val Ile Arg Cys Met Thr Asn Thr Pro Val Val Val Gln 115120 125 Glu Gly Ala Thr Val Tyr Ala Thr Gly Thr His Ala Leu Val Glu Asp130 135 140 Gly Gln Leu Leu Glu Gln Leu Met Ser Ser Val Gly Phe Cys ThrGlu 145 150 155 160 Val Glu Glu Asp Leu Ile Asp Ala Val Thr Gly Leu SerGly Ser Lys 165 170 175 Pro Ala Tyr Ala Phe Met Ala Leu Asp Ala Leu AlaAsp Gly Gly Val 180 185 190 Lys Met Gly Leu Pro Arg Arg Leu Ala Ile GlnLeu Gly Ala Gln Ala 195 200 205 Leu Leu Gly Ala Ala Lys Met Leu Leu AspSer Glu Gln His Pro Cys 210 215 220 Gln Leu Lys Asp Asn Val Cys Ser ProGly Gly Ala Thr Ile His Ala 225 230 235 240 Leu His Phe Leu Glu Ser GlyGly Phe Arg Ser Leu Leu Ile Asn Ala 245 250 255 Val Glu Ala Ser Cys IleArg Thr Arg Glu Leu Gln Ser Met Ala Asp 260 265 270 Gln Glu Lys Ile SerPro Ala Ala Leu Lys Lys Thr Leu Leu Asp Arg 275 280 285 Val Lys Leu GluSer Pro Thr Val Ser Thr Leu Thr Pro Ser Ser Pro 290 295 300 Gly Lys LeuLeu Thr Arg Ser Leu Ala Leu Gly Gly Lys Lys Asp 305 310 315

What is claimed is:
 1. A method of identifying a candidate p53 pathwaymodulating agent, said method comprising the steps of: (a) providing anassay system comprising a purified P5CR polypeptide or nucleic acid or afunctionally active fragment or derivative thereof; (b) contacting theassay system with a test agent under conditions whereby, but for thepresence of the test agent, the system provides a reference activity;and (c) detecting a test agent-biased activity of the assay system,wherein a difference between the test agent-biased activity and thereference activity identifies the test agent as a candidate p53 pathwaymodulating agent.
 2. The method of claim 1 wherein the assay systemcomprises cultured cells that express the P5CR polypeptide.
 3. Themethod of claim 2 wherein the cultured cells additionally have defectivep53 function.
 4. The method of claim 1 wherein the assay system includesa screening assay comprising a P5CR polypeptide, and the candidate testagent is a small molecule modulator.
 5. The method of claim 4 whereinthe assay is a reductase assay.
 6. The method of claim 1 wherein theassay system is selected from the group consisting of an apoptosis assaysystem, a cell proliferation assay system, an angiogenesis assay system,and a hypoxic induction assay system.
 7. The method of claim 1 whereinthe assay system includes a binding assay comprising a P5CR polypeptideand the candidate test agent is an antibody.
 8. The method of claim 1wherein the assay system includes an expression assay comprising a P5CRnucleic acid and the candidate test agent is a nucleic acid modulator.9. The method of claim 8 wherein the nucleic acid modulator is anantisense oligomer.
 10. The method of claim 8 wherein the nucleic acidmodulator is a PMO.
 11. The method of claim 1 additionally comprising:(d) administering the candidate p53 pathway modulating agent identifiedin (c) to a model system comprising cells defective in p53 function and,detecting a phenotypic change in the model system that indicates thatthe p53 function is restored.
 12. The method of claim 11 wherein themodel system is a mouse model with defective p53 function.
 13. A methodfor modulating a p53 pathway of a cell comprising contacting a celldefective in p53 function with a candidate modulator that specificallybinds to a P5CR polypeptide comprising an amino acid sequence selectedfrom group consisting of SEQ ID NOs: 10, 11, 12, 13, and 14 whereby p53function is restored.
 14. The method of claim 13 wherein the candidatemodulator is administered to a vertebrate animal predetermined to have adisease or disorder resulting from a defect in p53 function.
 15. Themethod of claim 13 wherein the candidate modulator is selected from thegroup consisting of an antibody and a small molecule.
 16. The method ofclaim 1, comprising the additional steps of: (d) providing a secondaryassay system comprising cultured cells or a non-human animal expressingP5CR, (e) contacting the secondary assay system with the test agent of(b) or an agent derived therefrom under conditions whereby, but for thepresence of the test agent or agent derived therefrom, the systemprovides a reference activity; and (f) detecting an agent-biasedactivity of the second assay system, wherein a difference between theagent-biased activity and the reference activity of the second assaysystem confirms the test agent or agent derived therefrom as a candidatep53 pathway modulating agent, and wherein the second assay detects anagent-biased change in the p53 pathway.
 17. The method of claim 16wherein the secondary assay system comprises cultured cells.
 18. Themethod of claim 16 wherein the secondary assay system comprises anon-human animal.
 19. The method of claim 18 wherein the non-humananimal mis-expresses a p53 pathway gene.
 20. A method of modulating p53pathway in a mammalian cell comprising contacting the cell with an agentthat specifically binds a P5CR polypeptide or nucleic acid.
 21. Themethod of claim 20 wherein the agent is administered to a mammaliananimal predetermined to have a pathology associated with the p53pathway.
 22. The method of claim 20 wherein the agent is a smallmolecule modulator, a nucleic acid modulator, or an antibody.
 23. Amethod for diagnosing a disease in a patient comprising: (a) obtaining abiological sample from the patient; (b) contacting the sample with aprobe for P5CR expression; (c) comparing results from step (b) with acontrol; (d) determining whether step (c) indicates a likelihood ofdisease.
 24. The method of claim 23 wherein said disease is cancer. 25.The method according to claim 24, wherein said cancer is a cancer asshown in Table 1 as having >25% expression level.